Isolated Bacterial Strain of the Genus Burkholderia and Pesticidal Metabolites Therefrom

ABSTRACT

A species of  Burkholderia  sp with no known pathogenicity to vertebrates but with pesticidal activity (e.g., plants, insects, fungi, weeds and nematodes) is provided. Also provided are natural products derived from a culture of said species and methods of controlling pests using said natural products.

PRIORITY CLAIM

This application is a continuation-in-part of application Ser. No.13/034,575, filed Feb. 24, 2011, the contents of which are incorporatedherein by reference. Application Ser. No. 13/034,575 claims priority toU.S. application Ser. No. 61/308,287, filed Feb. 25, 2010 and priorityto application Ser. No. 61/406,541, filed Oct. 25, 2010 under 35 U.S.C.119(e). The contents of U.S. application Ser. No. 61/308,287, filed Feb.25, 2010 and U.S. application Ser. No. 61/406,541, filed Oct. 25, 2010are herein incorporated by reference

TECHNICAL FIELD

Provided herein is a species of Burkholderia sp with no knownpathogenicity to vertebrates, such as mammals, fish and birds butpesticidal activity against plants, insects, fungi and nematodes. Alsoprovided are natural products derived from a culture of said species andmethods of controlling germination and growth of dicotyledenous,monocotyledonous and sedge weeds, modulating growth of fungi andcontrolling pests such as insects and nematodes using said naturalproducts.

BACKGROUND

Natural products are substances produced by microbes, plants, and otherorganisms. Microbial natural products offer an abundant source ofchemical diversity, and there is a long history of utilizing naturalproducts for pharmaceutical purposes. One such compound is FR901228isolated from Chromobacterium and has been found to be useful as anantibacterial agent and antitumor agent (see, for example, Ueda et al.,U.S. Pat. No. 7,396,665).

However, secondary metabolites produced by microbes have also beensuccessfully found to have uses for weed and pest control inagricultural applications (see, for example, Nakajima et al. 1991; Dukeet al., 2000; Lydon & Duke, 1999; Gerwick et al., U.S. Pat. No.7,393,812). Microbial natural products have been also successfullydeveloped into agricultural insecticides (see, for example, Salama etal. 1981; Thompson et al., 2000; Krieg et al. 1983). Sometimes, suchnatural products have been combined with chemical pesticides (see, forexample, Gottlieb, U.S. Pat. No. 4,808,207).

Burkholderia

The Burkholderia genus, β-subdivision of the proteobacteria, comprisesmore than 40 species that inhabit diverse ecological niches (Compant etal., 2008). The bacterial species in the genus Burkholderia areubiquitous organisms in soil and rhizosphere (Coenye and Vandamme, 2003;Parke and Gurian-Sherman, 2001). Traditionally, they have been known asplant pathogens, B. cepacia being the first one discovered andidentified as the pathogen causing disease in onions (Burkholder, 1950).Several Burkholderia species have developed beneficial interactions withtheir plant hosts (see, for example, Cabballero-Mellado et al., 2004,Chen et al., 2007). Some Burkholderia species have also been found to beopportunistic human pathogens (see, for example, Cheng and Currie, 2005and Nierman et al., 2004). Additionally, some Burkholderia species havebeen found to have potential as biocontrol products (see for example,Burkhead et al., 1994; Knudsen et al., 1987; Jansiewicz et al., 1988;Gouge et al., US Patent Application No. 2003/0082147; Parke et al., U.S.Pat. No. 6,077,505; Casida et al., U.S. Pat. No. 6,689,357; Jeddeloh etal., WO2001055398; Zhang et al., U.S. Pat. No. 7,141,407). Some speciesof in this genus have been effective in bioremediation to decontaminatepolluted soil or groundwater (see, for example, Leahy et al. 1996).Further, some Burkholderia species have been found to secrete a varietyof extracellular enzymes with proteolytic, lipolytic and hemolyticactivities, as well as toxins, antibiotics, and siderophores (see, forexample, Ludovic et al., 2007; Nagamatsu, 2001).

Oxazoles, Thiazoles and Indoles

Oxazoles, thiazoles and indoles are widely distributed in plants, algae,sponges, and microorganisms. A large number of natural products containone or more of the five-membered oxazole, thiazole and indolenucleus/moieties. These natural products exhibit a broad spectrum ofbiological activity of demonstrable therapeutic value. For example,bleomycin A (Tomohisa et al.), a widely prescribed anticancer drug,effects the oxidative degradation of DNA and uses a bithiazole moiety tobind its target DNA sequences (Vanderwall et al., 1997). Bacitracin(Ming et al., 2002), a thiazoline-containing peptide antibiotic,interdicts bacterial cell wall new biosynthesis by complexation withC55-bactoprenolpyrophosphate. Thiangazole (Kunze et al., 1993) containsa tandem array of one oxazole and three thiazolines and exhibitsantiviral activity (Jansen et al., 1992). Yet otheroxazole/thiazole-containing natural products such as thiostrepton(Anderson et al., 1970) and GE2270A (Selva et al., 1997) inhibittranslation steps in bacterial protein synthesis. More than 1000alkaloids with the indole skeleton have been reported frommicroorganisms. One-third of these compounds are peptides with massesbeyond 500 Da where the indole is tryptophan derived. The structuralvariety of the remaining two-thirds is higher, and their biologicalactivity seems to cover a broader range, including antimicrobial,antiviral, cytotoxic, insecticidal, antithrombotic, or enzyme inhibitoryactivity.

BRIEF SUMMARY

Provided herein is an isolated strain of a non-Burkholderia cepacia,non-Burkholderia plantari, non-Burkholderia gladioli, Burkholderia sp.which has the following characteristics:

-   -   a. Has a 16S rRNA gene sequence comprising a forward sequences        having at least 99.0% identity to the sequences set forth in SEQ        ID NO:8, 11 and 12 and a reverse sequence having at least 99.0%        identity to SEQ ID NO:9, 10, 13-15;    -   b. Has pesticidal, in particular, herbicidal, insecticidal,        fungicidal and nematicidal activity;    -   c. Produces at least one of the compounds selected from the        group consisting of:        -   (i) a compound having the following properties: (a) a            molecular weight of about 525-555 as determined by Liquid            Chromatography/Mass Spectroscopy (LC/MS); (b) ¹H NMR values            of 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22,            3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23, 1.74, 1.15, 1.12,            1.05, 1.02; (c) has ¹³C NMR values of 172.99, 172.93,            169.57, 169.23, 167.59, 130.74, 130.12, 129.93, 128.32,            73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90,            30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51 and (c) an            High Pressure Liquid Chromatography (HPLC) retention time of            about 10-15 minutes, on a reversed phase C-18 HPLC column            using a water:acetonitrile (CH₃CN) gradient;        -   (ii) a compound having an oxazolyl-indole structure            comprising at least one indole moiety, at least one oxazole            moiety, at least one substituted alkyl group and at least            one carboxylic ester group; at least 17 carbons and at least            3 oxygen and 2 nitrogens;        -   (iii) a compound having an oxazolyl-benzyl structure            comprising at least one benzyl moiety, at least one oxazole            moiety, at least one substituted alkyl group and at least            one amide group; at least 15 carbons and at least 2 oxygen            and 2 nitrogens;        -   (iv) a compound having at least one ester, at least one            amide, at least three methylene groups, at least one            tetrahydropyranose moiety and at least three olefinic double            bonds, at least six methyl groups, at least three hydroxyl            groups, at least twenty five carbons and at least eight            oxygen and one nitrogen and    -   d. is non-pathogenic (non-infectious) to vertebrate animals,        such as mammals, birds and fish;    -   e. is susceptible to kanamycin, chloramphenicol, ciprofloxacin,        piperacillin, imipenem, and a combination of sulphamethoxazole        and trimethoprim and    -   f. contains the fatty acids 16:0, cyclo 17:0, 16:0 3-OH, 14:0,        cyclo 19:0 ω8c, 18:0.

In a particular embodiment, the strain has the identifyingcharacteristics of a Burkholderia A396 strain (NRRL Accession No.B-50319).

Disclosed herein are isolated compounds which are optionally obtainableor derived from Burkholderia species, or alternatively, organismscapable of producing these compounds that can be used to control variouspests, particularly plant phytopathogenic pests, examples of whichinclude but are not limited to insects, nematodes, bacteria, fungi.These compounds may also be used as herbicides.

In particular, the isolated pesticidal compounds may include but are notlimited to:

(A) a compound having the following properties: (i) a molecular weightof about 525-555 as determined by Liquid Chromatography/MassSpectroscopy (LC/MS); (ii) ¹H NMR δ values of 6.22, 5.81, 5.69, 5.66,5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23,1.74, 1.15, 1.12, 1.05, 1.02; (iii) has ¹³C NMR δ values of 172.99,172.93, 169.57, 169.23, 167.59, 130.74, 130.12, 129.93, 128.32, 73.49,62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90, 30.36, 29.26, 18.59,18.38, 18.09, 17.93, 12.51 and (iv) an High Pressure LiquidChromatography (HPLC) retention time of about 10-15 minutes, on areversed phase C-18 HPLC column using a water:acetonitrile (CH₃CN)gradient;

(B) a compound having an oxazolyl-indole structure comprising at leastone indole moiety, at least one oxazole moiety, at least one substitutedalkyl group and at least one carboxylic ester group; at least 17 carbonsand at least 3 oxygen and 2 nitrogens;

(C) a compound having an oxazolyl-benzyl structure comprising at leastone benzyl moiety, at least one oxazole moiety, at least one substitutedalkyl group and at least one amide group; at least 15 carbons and atleast 2 oxygen and 2 nitrogens;

(D) a compound having at least one ester, at least one amide, at leastthree methylene groups, at least one tetrahydropyranose moiety and atleast three olefinic double bonds, at least six methyl groups, at leastthree hydroxyl groups, at least twenty five carbons and at least eightoxygen and one nitrogen and

(E) a compound having at least one ester, at least one amide, an epoxidemethylene group, at least one tetrahydropyranose moiety, at least threeolefinic double bonds, at least six methyl groups, at least threehydroxyl groups, at least 25 carbons, at least 8 oxygens and at least 1nitrogen.

In a particular embodiment, the isolated compounds may include but arenot limited to:

(A) a compound having an oxazolyl-indole structure comprising at leastone indole moiety, at least one oxazole moiety, at least one substitutedalkyl group, at least one carboxylic ester group, at least 17 carbons,at least 3 oxygens and at least 2 nitrogens; and which has at least oneof the following: (i) a molecular weight of about 275-435; (ii) ¹H NMR δvalues at 8.44, 8.74, 8.19, 7.47, 7.31, 3.98, 2.82, 2.33, 1.08; (iii)¹³C NMR δ values of δ 163.7, 161.2, 154.8, 136.1, 129.4, 125.4, 123.5,123.3, 121.8, 121.5, 111.8, 104.7, 52.2, 37.3, 28.1, 22.7, 22.7; (iv) anHigh Pressure Liquid Chromatography (HPLC) retention time of about 10-20minutes on a reversed phase C-18 HPLC column using a water:acetonitrile(CH₃CN) with a gradient solvent system and UV detection of 210 nm; (v)UV absorption bands at about 226, 275, 327 nm.;

(B) a compound having an oxazolyl-benzyl structure comprising at leastone benzyl moiety, at least one oxazole moiety, at least one substitutedalkyl group and at least one amide group; at least 15 carbons and atleast 2 oxygens, at least 2 nitrogens; and at least one of the followingcharacteristics: (i) a molecular weight of about 240-290 as determinedby Liquid Chromatography/Mass Spectroscopy (LC/MS); (ii)¹H NMR δ valuesat about 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93; (iii)¹³C NMR δvalues of 158.2, 156.3, 155.5, 132.6, 129.5, 129.5, 127.3, 121.8, 115.2,115.2, 41.2, 35.3, 26.7, 21.5, 21.5; (iv) a High Pressure LiquidChromatography (HPLC) retention time of about 6-15 minutes, on areversed phase C-18 HPLC column using a water:acetonitrile (CH₃CN)gradient and (v) UV absorption bands at about 230, 285, 323 nm;

(C) a non-epoxide compound comprising at least one ester, at least oneamide, at least three methylene groups, at least one tetrahydropyranosemoiety and at least three olefinic double bonds, at least six methylgroups, at least three hydroxyl groups, at least twenty five carbons, atleast eight oxygens and one nitrogen and at least one of the followingcharacteristics: (i) a molecular weight of about 530-580 as determinedby Liquid Chromatography/Mass Spectroscopy (LC/MS); (ii)¹H NMR δ valuesof 6.40, 6.39, 6.00, 5.97, 5.67, 5.54, 4.33, 3.77, 3.73, 3.70, 3.59,3.47, 3.41, 2.44, 2.35, 2.26, 1.97, 1.81, 1.76, 1.42, 1.37, 1.16, 1.12,1.04; (iii) ¹³C NMR δ values of 173.92, 166.06, 145.06, 138.76, 135.71,129.99, 126.20, 123.35, 99.75, 82.20, 78.22, 76.69, 71.23, 70.79, 70.48,69.84, 60.98, 48.84, 36.89, 33.09, 30.63, 28.55, 25.88, 20.37, 18.11,14.90, 12.81, 9.41; (iv) a High Pressure Liquid Chromatography (HPLC)retention time of about 7-12 minutes, on a reversed phase C-18 HPLCcolumn using a water:acetonitrile (CH₃CN) with a gradient solvent systemand UV detection of 210 nm; (v) a molecular formula of C₂₈H₄₅NO₁₀ whichwas determined by interpretation of the ESIMS and NMR data analysis;(vi) UV absorption bands between about 210-450 nm;

(D) a compound comprising (i) at least one ester, at least one amide, anepoxide methylene group, at least one tetrahydropyranose moiety and atleast three olefinic double bonds, at least six methyl groups, at leastthree hydroxyl groups, at least 25 carbons, at least 8 oxygens and atleast 1 nitrogen, (ii)¹³C NMR δ values of 174.03, 166.12, 143.63,137.50, 134.39, 128.70, 126.68, 124.41, 98.09, 80.75, 76.84, 75.23,69.87, 69.08, 68.69, 68.60, 48.83, 41.07, 35.45, 31.67, 29.19, 27.12,24.55, 19.20, 18.95, 13.48, 11.39, 8.04, (iii) a molecular formula ofC₂₈H₄₃NO₉ and at least one of: (i)¹H NMR δ values at about 6.41, 6.40,6.01, 5.97, 5.67, 5.55, 4.33, 3.77, 3.75, 3.72, 3.64, 3.59, 3.54, 3.52,2.44, 2.34, 2.25, 1.96, 1.81, 1.76, 1.42, 1.38, 1.17, 1.12, 1.04; (ii)an High Pressure Liquid Chromatography (HPLC) retention time of about6-15 minutes, on a reversed phase C-18 HPLC column using awater:acetonitrile (CH₃CN) gradient; (iii) UV absorption band betweenabout 210-450 nm and most particularly at about 234 nm.

In a more particular embodiment, provided are compounds including butnot limited to:

(A) a compound having the structure ##STR001##

or a pesticidally acceptable salt or steriosomers thereof, wherein M is1, 2, 3 or 4; n is 0, 1, 2, or 3; p and q are independently 1 or 2; X isO, NH or NR; R1, R2 and R3 are the same or different and independentlyan amino acid side-chain moiety or an amino acid side-chain derivativeand R is a lower chain alkyl, aryl or arylalkyl moiety;

(B) a compound having the structure ##STR002##

wherein X, Y and Z are each independently —O, —NR₁, or —S, wherein R₁ is—H or C₁-C₁₀ alkyl; R₁, R₂ and m are each independently —H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, cycloalkyl, substitutedcycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substitutedthioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl,oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl and “m” may belocated anywhere on the oxazole ring;

-   -   (C) a compound having the structure ##STR002a##

wherein R₁ is —H or C₁-C₁₀ alkyl; R₂ is an alkyl ester;

(D) a compound having the structure ##STR003##

wherein: X and Y are each independently —OH, —NR₁, or —S, wherein R₁ is—H or C₁-C₁₀ alkyl; R₁, R₂ and m, a substituent on the oxazole ring, areeach independently —H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl;

(E) a compound having the structure ##STR003a##

wherein R₁ is —H or C₁-C₁₀ alkyl;

(F) a compound having the structure ##STR004a##

Wherein X, Y and Z are each independently-0, —NR, or —S, wherein R is Hor C₁-C₁₀ alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, andR₁₃ are each independently H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.

(G) a compound having the structure ##STR004b##

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, and R₁₃ areeach independently H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl;

-   -   (H) a compound having the structure ##STR004c##

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₁₁, are each independently H,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl,substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl,substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;

(I) a compound having the structure ##STR005##

wherein X and Y are each independently —OH, —NR₁, or —S, wherein R₁, R₂are each independently —H, alkyl (e.g., C₁-C₁₀ alkyl), substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy,substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen,amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl;

(J) a compound having the structure ##STR006a##

Wherein X, Y and Z are each independently-0, —NR, or —S, wherein R is Hor C₁-C₁₀ alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₁₁, R₁₂, and R₁₃ areeach independently H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.

In a most particular embodiment, the compounds may include but are notlimited to

-   -   (i) templazole A;    -   (ii) templazole B;    -   (iii) templamide A;    -   (iv) templamide B;    -   (v) FR90128;

(XL) FR901465

Also provided are methods of obtaining the compounds set forth above. Inparticular, the method comprises culturing the Burkholderia straindisclosed herein and producing the compound. Further provided is amethod for isolating these compounds by isolating the compound(s)produced by a Burkholderia strain comprising isolating compoundsproduced from a supernatant of a culture of said Burkholderia strain.

Further provided is a combination comprising (a) a first substanceselected from the group consisting of (i) a pure culture, whole cellbroth, comprising or cell fraction, filtrate or supernatant derived fromthe Burkholderia strain set forth above or extract thereof for useoptionally as a pesticide; (ii) one or more of the compounds set forthabove (b) optionally a second substance, wherein said second substanceis a chemical or biological pesticide and (c) optionally at least one ofa carrier, diluent, surfactant, adjuvant, or pesticide. In a particularembodiment, the combination is a composition. In a related aspect,provided herein is a seed coated with said composition. The seed may bea genetically modified seed that is herbicide resistant.

In a related aspect, disclosed is a method for modulating pestinfestation in a plant comprising applying to the plant and/or seedsthereof and/or substrate used for growing said plant and/or a method formodulating emergence and/or growth of monocotyledonous, sedge ordicotyledonous weeds comprising applying to said weed or soil an amountof

-   -   (I) (a) the isolated compounds set forth above and (b)        optionally another substance, wherein said substance is a        pesticide (e.g. nematocide, herbicide, fungicide, insecticide)        or    -   (II) the composition or combination set forth above        in an amount effective to modulate pest infestation and/or        emergence or growth of monocotyledonous, sedge or dicotyledonous        weeds.

In another related aspect, provided is the use of the strains, cultures,extracts, supernatants, combinations, compounds set forth above formodulating pest infestation in a plant comprising applying to the plantand/or seeds thereof and/or substrate used for growing said plant and/ora method for modulating emergence and/or growth of monocotyledonous,sedge or dicotyledonous weeds. The weeds may be grass weeds (e.g.,Digitaria sanguinalis, Echinochloa grus-gali, Phalaris minor and Loliumperenne), sedge weeds (e.g., Cyperus difformis) or broadleaf weeds(e.g., Brassica juncea, Trifolium repens, Conyza canadensis, Conyzabonariensis, Amaranthus palmeri, Amaranthus rudis, Ambrosiaartemisifolia, Ambrosia trifida, Kochia scoparia, Solanum nigrum, Oxalisstricta, Chenopodium album, Medicago polymorpha, Taraxacum oficinale,Convolvulus arvensos, Pueraria lobata, Malva parviflora, Galliumaparine). Further provided are seeds coated with the combinations,cultures, extracts, strains, compounds supernatant, whole cell broth,cell fractions set forth above. The seeds may be genetically modifiedseeds that may be herbicide resistant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the comparison of the growth rate of Burkholderia A396 toBurkholderia multivorans ATCC 17616.

FIG. 2 shows the effect of Burkholderia A396 extract on bindweed.

FIG. 3 shows the effect of Burkholderia A396 extract on pigweed.

FIG. 4 shows the effect of Burkholderia A396 extract on Cabbage looper(Tricoplusia ni).

FIG. 5 shows the effect of Burkholderia A396 culture broth on Beetarmyworm (Spodoptera exigua).

FIG. 6 shows the effect of Burkholderia A396 culture broth on themotility of juvenile root-knot nematodes (Meloidogyne incognita).

FIG. 7 is a schematic representation of purification scheme forobtaining the templazole and templamide compounds.

FIG. 8 shows results of an in vitro assay to test the fungicidal effectof FR90128 on Botrytis cinerea (left) and Phytophtora sp. (right).

FIG. 9 shows the effect of Burkholderia A396 culture broth on theaverage gall index (% control) of cucumber roots cv. Toschka inoculatedwith 3000 eggs of Meloidogyne sp. 14 days after inoculation andapplication.

FIG. 10 Effect of Burkholderia A396 culture broth on the average gallindex of cucumber roots cv. Toschka inoculated with 3000 eggs ofMeloidogyne sp. 14 days after inoculation and application.

DETAILED DESCRIPTION OF EMBODIMENTS

While the compositions and methods heretofore are susceptible to variousmodifications and alternative forms, exemplary embodiments will hereinbe described in detail. It should be understood, however, that there isno intent to limit the invention to the particular forms disclosed, buton the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is included therein. Smaller ranges are also included. Theupper and lower limits of these smaller ranges are also includedtherein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” and and the include plural references unless thecontext clearly dictates otherwise.

As defined herein, “derived from” means directly isolated or obtainedfrom a particular source or alternatively having identifyingcharacteristics of a substance or organism isolated or obtained from aparticular source.

As defined herein, an “isolated compound” is essentially free of othercompounds or substances, e.g., at least about 20% pure, preferably atleast about 40% pure, more preferably about 60% pure, even morepreferably about 80% pure, most preferably about 90% pure, and even mostpreferably about 95% pure, as determined by analytical methods,including but not limited to chromatographic methods, electrophoreticmethods.

As used herein, the term “alkyl” refers to a monovalent straight orbranched chain hydrocarbon group having from one to about 12 carbonatoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

As used herein, “substituted alkyl” refers to alkyl groups furtherbearing one or more substituents selected from hydroxy, alkoxy,mercapto, cycloalkyl, substituted cycloalkyl, heterocyclic, substitutedheterocyclic, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, aryloxy, substituted aryloxy, halogen, cyano, nitro, amino,amido, —C(O)H, acyl, oxyacyl, carboxyl, sulfonyl, sulfonamide, sulfuryl,and the like.

As used herein, “alkenyl” refers to straight or branched chainhydrocarbyl groups having one or more carbon-carbon double bonds, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkenyl” refers to alkenyl groups further bearing one or moresubstituents as set forth above.

As used herein, “alkynyl” refers to straight or branched chainhydrocarbyl groups having at least one carbon-carbon triple bond, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkynyl” refers to alkynyl groups further bearing one or moresubstituents as set forth above.

As used herein, “aryl” refers to aromatic groups having in the range of6 up to 14 carbon atoms and “substituted aryl” refers to aryl groupsfurther bearing one or more substituents as set forth above.

As used herein, “heteroaryl” refers to aromatic rings containing one ormore heteroatoms (e.g., N, O, S, or the like) as part of the ringstructure, and having in the range of 3 up to 14 carbon atoms and“substituted heteroaryl” refers to heteroaryl groups further bearing oneor more substituents as set forth above.

As used herein, “alkoxy” refers to the moiety —O-alkyl-, wherein alkylis as defined above, and “substituted alkoxy” refers to alkoxyl groupsfurther bearing one or more substituents as set forth above.

As used herein, “thioalkyl” refers to the moiety —S-alkyl-, whereinalkyl is as defined above, and “substituted thioalkyl” refers tothioalkyl groups further bearing one or more substituents as set forthabove.

As used herein, “cycloalkyl” refers to ring-containing alkyl groupscontaining in the range of about 3 up to 8 carbon atoms, and“substituted cycloalkyl” refers to cycloalkyl groups further bearing oneor more substituents as set forth above.

As used herein, “heterocyclic”, refers to cyclic (i.e., ring-containing)groups containing one or more heteroatoms (e.g., N, O, S, or the like)as part of the ring structure, and having in the range of 3 up to 14carbon atoms and “substituted heterocyclic” refers to heterocyclicgroups further bearing one or more substituent's as set forth above.

The Burkholderia Strain

The Burkholderia strain set forth herein is a non-Burkholderia cepaciacomplex, non-Burkholderia plantari, non-Burkholderia gladioli,Burkholderia sp and non-pathogenic to vertebrates, such as birds,mammals and fish. This strain may be isolated from a soil sample usingprocedures known in the art and described by Lorch et al., 1995. TheBurkholderia strain may be isolated from many different types of soil orgrowth medium. The sample is then plated on potato dextrose agar (PDA).The bacteria are gram negative, and it forms round, opaque cream-coloredcolonies that change to pink and pinkish-brown in color and mucoid orslimy over time.

Colonies are isolated from the potato dextrose agar plates and screenedfor those that have biological, genetic, biochemical and/or enzymaticcharacteristics of the Burkholderia strain of the present invention setforth in the Examples below. In particular, the Burkholderia strain hasa 16S rRNA gene comprising a forward sequence that is at least about99.0%, preferably about 99.5%, more preferably about 99.9% and mostpreferably about 100% identical to the sequence set forth in SEQ ID NO:8, 11 and 12 and a forward sequence that is at least about 99.0%,preferably about 99.5%, more preferably about 99.9% and most preferablyabout 100% identical to the sequence set forth in SEQ ID NO: 9, 10, 13,14 and 15 as determined by clustal analysis. Furthermore, as set forthbelow, this Burkholderia strain may, as set forth below, have pesticidalactivity, particularly, virucidal, herbicidal, germicidal, fungicidal,nematicidal, bactericidal and insecticidal and more particularly,herbicidal, insecticidal, fungicidal and nematicidal activity. It is notpathogenic to vertebrate animals, such as mammals, birds, and fish.

Additionally, the Burkholderia strain produces at least the pesticidalcompounds set forth in the instant disclosure.

The Burkholderia strain is susceptible to kanamycin, chloramphenicol,ciprofloxacin, piperacillin, imipenem, and a combination ofsulphamethoxazole and trimethoprim and contains the fatty acids 16:0,cyclo 17:0, 16:0 3-OH, 14:0, cyclo 19:0, 18:0.

This Burkholderia strain may be obtained by culturing a microorganismhaving the identifying characteristics of Burkholderia A396 (NRRLAccession No. B-50319) on Potato Dextrose Agar (PDA) or in afermentation medium containing defined carbon sources such as glucose,maltose, fructose, galactose, and undefined nitrogen sources such aspeptone, tryptone, soytone, and NZ amine.

Pesticidal Compounds

The pesticidal compound disclosed herein may have the followingproperties: (a) is obtainable from a novel Burkholderia species, e.g.,A396; (b) is, in particular, toxic to most common agricultural insectpests; (c) has a molecular weight of about 525-555 and moreparticularly, 540 as determined by Liquid Chromatography/MassSpectroscopy (LC/MS); (d) has ¹H NMR values of 6.22, 5.81, 5.69, 5.66,5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23,1.74, 1.15, 1.12, 1.05, 1.02; (d) has ¹³C NMR values of 172.99, 172.93,169.57, 169.23, 167.59, 130.74, 130.12, 129.93, 128.32, 73.49, 62.95,59.42, 57.73, 38.39, 38.00, 35.49, 30.90, 30.36, 29.26, 18.59, 18.38,18.09, 17.93, 12.51 (e) has an High Pressure Liquid Chromatography(HPLC) retention time of about 10-15 minutes, more specifically about 12minutes and even more specifically about 12.14 min on a reversed phaseC-18 HPLC (Phenomenex, Luna 5μ C18 (2) 100 A, 100×4.60 mm) column usinga water:acetonitrile (CH₃CN) with a gradient solvent system (0-20 min90-0% aqueous CH₃CN, 20-24 min 100% CH₃CN, 24-27 min, 0-90% aqueousCH₃CN, 27-30 min 90% aqueous CH₃CN) at 0.5 mL/min flow rate and UVdetection of 210 nm (f) has a molecular formula, C₂₄H₃₆N₄O₆S₂, which isdetermined by interpretation of ¹H, ¹³C NMR and LC/MS data (g) a ¹³C NMRspectrum with signals for all 24 carbons, including 5 methyl, 4methylene, 9 methine, and 6 quaternary carbons and (g) ¹H NMR spectrumdisplaying characteristics of a typical depsipeptide, illustratingthree-amino protons [4.63, 4.31, 3.93], and one ester carbinol proton[5.69]. In a particular embodiment, the compound has the structure##STR001##:

Or a pesticidally acceptable salt or stereoisomers thereof, wherein M is1, 2, 3 or 4; n is 0, 1, 2, or 3; p and q are independently 1 or 2; X isO, NH or NR; R1, R2 and R3 are the same or different and independentlyan amino acid side-chain moiety or an amino acid side-chain derivativeand R is a lower chain alkyl, aryl or arylalkyl moiety.

In an even more particular embodiment, the compound has the structure ofFR90128:

Provided herewith are compounds set forth in ##STR002##:

wherein: X, Y and Z are each independently —O, —NR₁, or —S, wherein R₁is —H or C₁-C₁₀ alkyl; R₁, R₂ and m are each independently —H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, cycloalkyl, substitutedcycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substitutedthioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl,oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

In an even another particular embodiment, Family ##STR002## compoundsmay be the compounds set forth in (vi)-(xix).

These are from either natural materials or compounds obtained fromcommercial sources or by chemical synthesis. Natural sources of Family##STR002## compounds include, but are not limited to, microorganisms,alga, and sponges. In a more particular embodiment, microorganisms whichinclude the Family ##STR002## compounds include but are not limited to,or alternatively, Family ##STR002## compounds may be derived fromspecies such as Streptoverticillium waksmanii (compound vi) (Umehara, etal., 1984), Streptomyces pimprina (compound vii) (Naik et al., 2001),Streptoverticillium olivoreticuli (compounds viii, ix, x) (Koyama Y., etal., 1981), Streptomyces sp (compounds xi, xii) (Watabe et al., 1988),Pseudomonas syringae (compounds xiii, xiv) (Pettit et al., 2002). Family##STR002## compounds may also be derived from algae including but notlimited to red alga (compound xv) (N'Diaye, et al., 1996), red algaMartensia fragilis (compound xvi) (Takahashi S. et al., 1998), Diazonachinensis (compounds xvii & xviii) (Lindquist N. et al., 1991),Rhodophycota haraldiophyllum sp (compound xix) (Guella et al., 1994).

Also provided is ##STR003##:

wherein: X and Y are each independently —OH, —NR₁, or —S, wherein R₁ is—H or C₁-C₁₀ alkyl; R₁, R₂ and m, a substituent on the oxazole ring, areeach independently —H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.

Further provided is ##STR005##:

wherein X and Y are each independently —OH, —NR₁, or —S, wherein R₁, R₂are each independently —H, alkyl (e.g., C₁-C₁₀alkyl), substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy,substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen,amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.

In a particular embodiment, Family ##STR005## compounds such ascompounds from xx-xxiii set forth below may be derived from natural orcommercial sources or by chemical synthesis:

Natural sources of Family ##STR005## compounds include, but are notlimited to plants, corals, microorganisms, and sponges. Themicroorganisms include, but are not limited to Streptomyces griseus(compound xx) (Hirota et al., 1978), Streptomyces albus (compound xxi)(Werner et al., 1980). Family STR004 compounds may also be derived fromalgae including but not limited to Haraldiophyllum sp (compound xxii(Guella et al., 2006), and red algae (compound xxiii) (N'Diaye et al.,1994).

In one embodiment, the compound may be derived from or is obtainablefrom a microorganism, and in particular from Burkholderia species andcharacterized as having a structure comprising at least one ester, atleast one amide, at least three methylene groups, at least onetetrahydropyranose moiety and at least three olefinic double bonds, atleast six methyl groups, at least three hydroxyl groups, at least twentyfive carbons and at least eight oxygen and one nitrogen. The compoundfurther comprises at least one of the following characteristics:

(a) pesticidal properties and in particular, nematicidal, fungicidal,insecticidal and herbicidal properties;

(b) a molecular weight of about 530-580 and more particularly, 555 asdetermined by Liquid Chromatography/Mass Spectroscopy (LC/MS);

(c) ¹H NMR values of δ 6.40, 6.39, 6.00, 5.97, 5.67, 5.54, 4.33, 3.77,3.73, 3.70, 3.59, 3.47, 3.41, 2.44, 2.35, 2.26, 1.97, 1.81, 1.76, 1.42,1.37, 1.16, 1.12, 1.04;

(d)¹³C NMR values of δ 173.92, 166.06, 145.06, 138.76, 135.71, 129.99,126.20, 123.35, 99.75, 82.20, 78.22, 76.69, 71.23, 70.79, 70.48, 69.84,60.98, 48.84, 36.89, 33.09, 30.63, 28.55, 25.88, 20.37, 18.11, 14.90,12.81, 9.41;

(e) an High Pressure Liquid Chromatography (HPLC) retention time ofabout 7-12 minutes, more specifically about 10 minutes and even morespecifically about 10.98 min on a reversed phase C-18 HPLC (Phenomenex,Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile(CH₃CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN,20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90%aqueous CH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm;

(f) ¹³C NMR spectrum which exhibits 28 discrete carbon signals which maybe attributed to six methyls, four methylene carbons, and thirteenmethines including five sp², four quaternary carbons;

(g) a molecular formula of C₂₈H₄₅NO₁₀ which was determined byinterpretation of the ESIMS and NMR data analysis;

(h) UV absorption bands between about 210-450 nm and most particularlyat about 234 nm.

Also provided are compounds having the structure ##STR004a##:

Wherein X, Y and Z are each independently-0, —NR, or —S, wherein R is Hor C₁-C₁₀alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, andR₁₃ are each independently H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.

In a particular embodiment, the compound has the structure set forth in##STR004b##:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, and R₁₃ areas previously defined for ##STR004a##.

In a more particular embodiment, the compound is Templamide A with thefollowing structure:

In another embodiment, provided is a compound having formula##STR004c##:

Wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₁₁ are as previouslydefined for ##STR004a##.

In another embodiment, provided is a compound which may be derived fromBurkholderia species and characterized as having a structure comprisingat least one ester, at least one amide, an epoxide methylene group, atleast one tetrahydropyranose moiety and at least three olefinic doublebonds, at least six methyl groups, at least three hydroxyl groups, atleast 25 carbons and at least 8 oxygen and 1 nitrogen, and pesticideactivity. The compound further comprises at least one of the followingcharacteristics:

(a) pesticidal properties and in particular, insecticidal, fungicidal,nematocidal and herbicidal properties;

(b) a molecular weight of about 520-560 and particularly 537 asdetermined by Liquid Chromatography/Mass Spectroscopy (LC/MS);

(c)¹H NMR δ values at about 6.41, 6.40, 6.01, 5.97, 5.67, 5.55, 4.33,3.77, 3.75, 3.72, 3.64, 3.59, 3.54, 3.52, 2.44, 2.34, 2.25, 1.96, 1.81,1.76, 1.42, 1.38, 1.17, 1.12, 1.04;

(d)¹³C NMR values of δ 174.03, 166.12, 143.63, 137.50, 134.39, 128.70,126.68, 124.41, 98.09, 80.75, 76.84, 75.23, 69.87, 69.08, 68.69, 68.60,48.83, 41.07, 35.45, 31.67, 29.19, 27.12, 24.55, 19.20, 18.95, 13.48,11.39, 8.04;

(e) High Pressure Liquid Chromatography (HPLC) retention time of about6-15 minutes, more specifically about 8 minutes on a reversed phase C-18HPLC column using a water:acetonitrile (CH₃CN) gradient, particularly,an High Pressure Liquid Chromatography (HPLC) retention time of about8-15 minutes, more specifically about 11 minutes and even morespecifically about 11.73 min on a reversed phase C-18 HPLC (Phenomenex,Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile(CH₃CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN,20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90%aqueous CH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm;

(f) a molecular formula of C₂₈H₄₃NO₉ which was determined byinterpretation of the ESIMS and NMR data analysis;

(g) UV absorption bands at about 210-450 nm and most particularly atabout 234 nm.

In a particular embodiment, the compound has the structure ##STR006a##:

Wherein X, Y and Z are each independently-0, —NR, or —S, wherein R is Hor C₁-C₁₀ alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₁₁, R₁₂, and R₁₃ areeach independently H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.

In a particular embodiment, the compound has the structure:

In another embodiment, provided is a compound having formula##STR006b##:

Wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₁₁ are as previouslydefined for ##STR006a##.

In a more particular embodiment, the compound is Templamide B with thefollowing structure:

In yet another particular embodiment, the compound may be derived fromBurkholderia species and characterized as having a structure comprisingat least one ester, at least one amide, an epoxide methylene group, atleast one tetrahydropyranose moiety and at least three olefinic doublebonds, at least six methyl groups, at least three hydroxyl groups, atleast 25 carbons and at least 8 oxygens and at least 1 nitrogen. Thecompound further comprises at least one of the followingcharacteristics:

(a) pesticidal properties and in particular, insecticidal, fungicidal,nematicidal and herbicidal properties;

(b) a molecular weight of about 510-550 and particularly about 523 asdetermined by Liquid Chromatography/Mass Spectroscopy (LC/MS);

(c) ¹H NMR δ values at about 6.41, 6.40, 6.01, 5.98, 5.68, 5.56, 4.33,3.77, 3.75, 3.72, 3.65, 3.59, 3.55, 3.50, 2.44, 2.26, 2.04, 1.96, 1.81,1.75, 1.37, 1.17, 1.04;

(d) ¹³C NMR δ values of 172.22, 167.55, 144.98, 138.94, 135.84, 130.14,125.85, 123.37, 99.54, 82.19, 78.28, 76.69, 71.31, 70.13, 69.68, 48.83,42.52, 36.89, 33.11, 30.63, 25.99, 21.20, 20.38, 18.14, 14.93, 12.84;

(e) an High Pressure Liquid Chromatography (HPLC) retention time ofabout 6-15 minutes, more specifically about 8 minutes on a reversedphase C-18 HPLC column using a water:acetonitrile (CH₃CN) gradient,particularly, an High Pressure Liquid Chromatography (HPLC) retentiontime of about 8-15 minutes, more specifically about 10 minutes and evenmore specifically about 10.98 min on a reversed phase C-18 HPLC(Phenomenex, Luna 5μ C18(2) 100 A, 100×4.60 mm) column using awater:acetonitrile (CH₃CN) with a gradient solvent system (0-20 min;90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueousCH₃CN, 27-30 min; 90% aqueous CH₃CN) at 0.5 mL/min flow rate and UVdetection of 210 nm;

(f) a molecular formula of C₂₇H₄₁NO₉ which was determined byinterpretation of the ESIMS and NMR data analysis;

-   -   (g) UV absorption bands at about 210-450 nm and most        particularly at about 234 nm.

In a more particular embodiment, the compound is a known compoundFR901465 which was isolated earlier from culture broth of a bacterium ofPseudomonas sp. No. 2663 (Nakajima et al. 1996) and had been reported tohave anticancer activity with the following structure:

In an even another particular embodiment, Family ##STR006a## compoundsmay be the compounds set forth in xxiv to xxxix. These are from eithernatural materials or compounds obtained from commercial sources or bychemical synthesis. Natural sources of Family ##STR006a## compoundsinclude, but are not limited to, microorganisms, alga, and sponges. In amore particular embodiment, microorganisms which include the Family##STR006a## compounds which may be derived from species such asPseudomonas sp. No. 2663 (compounds xxiv-xxvi) (Nakajima et al., 1996).The synthetic analogues of the FR901464 (xxvii-xxxix) which have beensynthesized and patented as anticancer compounds (see Koide et al., USPatent Application No. 2008/0096879 A1).

Compositions

A substantially pure culture, cell fraction or supernatant and compoundsproduced by the Burkholderia strain of the present invention, may beformulated into pesticidal compositions.

The substances set forth above can be formulated in any manner.Non-limiting formulation examples include but are not limited toemulsifiable concentrates (EC), wettable powders (WP), soluble liquids(SL), aerosols, ultra-low volume concentrate solutions (ULV), solublepowders (SP), microencapsulation, water dispersed granules, flowables(FL), microemulsions (ME), nano-emulsions (NE), etc. In particular, theconcentrate, powders, granules and emulsions may be freeze-dried. In anyformulation described herein, percent of the active ingredient is withina range of 0.01% to 99.99%.

The compositions may be in the form of a liquid, gel or solid. Liquidcompositions comprise pesticidal compounds derived from saidBurkholderia strain, e.g. a strain having the identifyingcharacteristics of Burkholderia A396 (NRRL Accession No. B-50319).

A solid composition can be prepared by suspending a solid carrier in asolution of pesticidal compounds and drying the suspension under mildconditions, such as evaporation at room temperature or vacuumevaporation at 65° C. or lower.

A composition of the invention may comprise gel-encapsulated compoundsderived from the Burkholderia strain of the present invention. Suchgel-encapsulated materials can be prepared by mixing a gel-forming agent(e.g., gelatin, cellulose, or lignin) with a solution of pesticidalcompounds used in the method of the invention; and inducing gelformation of the agent.

The composition may additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)monolaurate. The concentration of surfactants may range between 0.1-35%of the total formulation, preferred range is 5-25%. The choice ofdispersing and emulsifying agents, such as non-ionic, anionic,amphoteric and cationic dispersing and emulsifying agents, and theamount employed is determined by the nature of the composition and theability of the agent to facilitate the dispersion of these compositions.

The composition may further comprise another microorganism and/orpesticide (e.g, nematocide, fungicide, insecticide). The microorganismmay include but is not limited to an agent derived from Bacillus sp.,Pseudomonas sp., Brevabacillus sp Lecanicillium sp., non-Ampelomycessp., Pseudozyma sp., Streptomyces sp, Burkholderia sp, Trichoderma sp,Gliocladium sp. Alternatively, the agent may be a natural oil oroil-product having fungicidal and/or insecticidal activity (e.g.,paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil,citrus oil, rosemary oil).

The composition, in particular, may further comprise an insecticide. Theinsecticide may include but is not limited to avermectin, Bacillusthuringiensis, neem oil and azadiractin, spinosads, Chromobacteriumsubtsugae, eucalyptus extract, entomopathogenic bacterium or fungi sucha Beauveria bassiana, and Metarrhizium anisopliae and chemicalinsecticides including but not limited to organochlorine compounds,organophosphorous compounds, carbamates, pyrethroids, andneonicotinoids.

The composition my further comprise a nematicide. The nematicide mayinclude, but is not limited to chemical nematicides such as fenamiphos,aldicarb, oxamyl, carbofuran, natural product neamticide, avermectin,the fungi Paecilomyces lilacinas and Muscodor spp., the bacteriaBacillus firmus and other Bacillus spp. and Pasteuria penetrans.

The composition may further comprise a biofungicide such as extract ofR. sachalinensis (Regalia) or a fungicide. Such fungicides include, butare not limited to, a single site anti-fungal agent which may includebut is not limited to benzimidazole, a demethylation inhibitor (DMI)(e.g., imidazole, piperazine, pyrimidine, triazole), morpholine,hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outsideinhibitor, quinoline, dicarboximide, carboximide, phenylamide,anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid,hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid(xylylalanine). In yet a further embodiment, the antifungal agent is ademethylation inhibitor selected from the group consisting of imidazole(e.g., triflumizole), piperazine, pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole, propiconazole, triadimefon,bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole,tebuconazole, tetraconazole, propiconazole).

The antimicrobial agent may also be a multi-site non-inorganic, chemicalfungicide selected from the group consisting of a nitrile (e.g.,chloronitrile or fludioxonil), quinoxaline, sulphamide, phosphonate,phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine,cyano-acetamide oxime.

The compositions may be applied using methods known in the art.Specifically, these compositions may be applied to plants or plantparts. Plants are to be understood as meaning in the present context allplants and plant populations such as desired and undesired wild plantsor crop plants (including naturally occurring crop plants). Crop plantscan be plants which can be obtained by conventional plant breeding andoptimization methods or by biotechnological and genetic engineeringmethods or by combinations of these methods, including the transgenicplants and including the plant cultivars protectable or not protectableby plant breeders' rights. Plant parts are to be understood as meaningall parts and organs of plants above and below the ground, such asshoot, leaf, flower and root, examples which may be mentioned beingleaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds,roots, tubers and rhizomes. The plant parts also include harvestedmaterial, and vegetative and generative propagation material, forexample cuttings, tubers, rhizomes, offshoots and seeds.

Treatment of the plants and plant parts with the compositions set forthabove may be carried out directly or by allowing the compositions to acton their surroundings, habitat or storage space by, for example,immersion, spraying, evaporation, fogging, scattering, painting on,injecting. In the case that the composition is applied to a seed, thecomposition may be applied to the seed as one or more coats prior toplanting the seed using one or more coats using methods known in theart.

As noted above, the compositions may be herbicidal compositions. Thecomposition may further comprise one or more herbicides. These mayinclude, but are not limited to, a bioherbicide and/or a chemicalherbicide. The bioherbicide may be selected from the group consisting ofclove, cinnamon, lemongrass, citrus oils, orange peel oil, tentoxin,cornexistin, AAL-toxin, leptospermone, thaxtomin, sarmentine,momilactone B, sorgoleone, ascaulatoxin and ascaulatoxin aglycone. Thechemical herbicide may include, but is not limited to, diflufenzopyr andsalts thereof, dicamba and salts thereof, topramezone, tembotrione,S-metolachlor, atrazine, mesotrione, primisulfuron-methyl,2,4-dichlorophenoxyacetic acid, nicosulfuron, thifensulfuron-methyl,asulam, metribuzin, diclofop-methyl, fluazifop, fenoxaprop-p-ethyl,asulam, oxyfluorfen, rimsulfuron, mecoprop, and quinclorac, thiobencarb,clomazone, cyhalofop, propanil, bensulfuron-methyl, penoxsulam,triclopyr, imazethapyr, halosulfuron-methyl, pendimethalin,bispyribac-sodium, carfentrazone ethyl, sodium bentazon/sodiumacifluorfen, glyphosate, glufosinate and orthosulfamuron.

Herbicidal compositions may be applied in liquid or solid form aspre-emergence or post-emergence formulations.

For pre-emergence dry formulations, the granule size of the carrier istypically 1-2 mm (diameter) but the granules can be either smaller orlarger depending on the required ground coverage. Granules may compriseporous or non-porous particles.

For post-emergence formulations, the formulation components used maycontain smectite clays, attapulgite clays and similar swelling clays,thickeners such as xanthan gums, gum Arabic and other polysaccharidethickeners as well as dispersion stabilizers such as nonionicsurfactants (for example polyoxyethylene (20) monolaurate).

Uses

The compositions and pesticidal compounds derived from the Burkholderiastrain set forth herein may be used as pesticides, particularly asinsecticides, nematocides, fungicides and herbicides.

Specifically, nematodes that may be controlled using the method setforth above include but are not limited to parasitic nematodes such asroot-knot, ring, sting, lance, cyst, and lesion nematodes, including butnot limited to Meloidogyne, Heterodera and Globodera spp; particularlyMeloidogyne incognita (root knot nematodes), as well as Globoderarostochiensis and globodera pailida (potato cyst nematodes); Heteroderaglycines (soybean cyst nematode); Heterodera schachtii (beet cystnematode); and Heterodera avenae (cereal cyst nematode).

Phytopathogenic insects controlled by the method of the presentinvention include but are not limited to insects from the order (a)Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp.,Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis,Archips spp., Argyrotaenia spp., Autographa spp., Busseola fusca, Cadracautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysiaambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp.,Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydiaspp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp.,Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp.,Ostrinia nubilalis, Pammene spp., Pandemis spp., Panolis flammea,Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp.,Tortrix spp., Trichoplusia ni and Yponomeuta spp.;

(b) Coleoptera, for example, Agriotes spp., Anthonomus spp., Atomarialinearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp.,Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp.,Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp.,Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp.,Psylliodes spp., Rhizopertha spp-, Scarabeidae, Sitophilus spp.,Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.; (c)Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp.,Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocercaspp.; (d) Isoptera, for example, Reticulitermes spp.; (e) Psocoptera,for example, Liposcelis spp.; (f) Anoplura, for example, Haematopinusspp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxeraspp.; (g) Mallophaga, for example, Damalinea spp. and Trichodectes spp.;(h) Thysanoptera, for example, Frankliniella spp., Hercinotnrips spp.,Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothripsaurantii; (i) Heteroptera, for example, Cimex spp., Distantiellatheobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisaspp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis,Scotinophara spp. and Tniatoma spp.; (j) Homoptera, for example,Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri; (k) Hymenoptera, for example, Acromyrmex, Atta spp.,Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampaspp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsisspp. and Vespa spp.; (l) Diptera, for example, Aedes spp., Antherigonasoccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp.,Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophilamelanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypodermaspp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp.,Musca spp., Oestrus spp., Orseolia spp., Oscinella fit, Pegomyiahyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxysspp., Tabanus spp., Tannia spp. and Tipula spp.; (m) Siphonaptera, forexample, Ceratophyllus spp. and Xenopsylla cheopis and (n) from theorder Thysanura, for example, Lepisma saccharina. The active ingredientsaccording to the invention may further be used for controlling cruciferflea beetles (Phyllotreta spp.), root maggots (Delia spp.), cabbageseedpod weevil (Ceutorhynchus spp.) and aphids in oil seed crops such ascanola (rape), mustard seed, and hybrids thereof, and also rice andmaize.

In a particular embodiment, the insect may be a member of theSpodoptera, more particularly, Spodoptera exigua, Myzus persicae,Plutella xylostella or Euschistus sp.

The substances and compositions may also be used to modulate emergencein either a pre-emergent or post-emergent formulation ofmonocotyledonous, sedge or dicotyledonous weeds. In a particularembodiment, the weeds may be

Chenopodium sp. (e.g., Chenopodium album, Chenopodium murale), Abutilonsp. (e.g., Abutilon theophrasti), Helianthus sp. (e.g., Helianthusannuus), Ambrosia sp. (e.g., Ambrosia artemesifolia, Ambrosia trifida),Amaranthus sp. (e.g., Amaranthus retroflexus, Amaranthus palmeri,Amaranthus rudis, Amaranthus spinosus, Amaranthus tuberculatus),Convolvulus sp.

(e.g., Convolvulus arvensis), Brassica sp. (e.g., Brassica kaber),Taraxacum sp. (e.g., Taraxacum officinale), Solanum sp. (e.g., Solanumnigrum, Solanum elaeagnifolium, Solanum physalifolium, Solanumptycanthum), Malva sp. (e.g., Malva neglecta, Malva parviflora), Setariasp. (e.g., Setaria lutescens), Bromus sp. (e.g., Bromus tectorum, Bromusdiandrus, Bromus hordeaceus), Poa sp. (e.g., Poa annua, Poa pratensis),Lolium sp. (e.g., Lolium perenne, Lolium rigidum, Lolium multiflorum L.var. Pace), Festuca sp. (e.g., Festuca arundinaceae, Festuca rubra),Echinochloa sp. (e.g., Echinochloa crus-galli, Echinochloa colona),Oxalis sp. (e.g., Oxalis stricta, Oxalis pes-caprae, Oxaliscorniculata); Cyperus sp. (e.g., Cyperus difformis, Cyperus esculentum,Cyperus rotundus, Cyperus brevifolius); Conyza sp. (e.g., Conyzacanadensis, Conyza sumatrensis, Conyza bonariensis); Sagina sp. (e.g.,Sagina procumbens); Pueraria lobata, Veronica sp. (e.g., Veronicahederafolia), Stellaria sp. (e.g., Stellaria media), Rorippa sp. (e.g.,Rorippa islandica), Senecio sp. (e.g., Senecio vulgaris), Lamium sp.(e.g., Lamium amplexicaule), Digitaria sp. (e.g., Digitaria sanguinalis,Digitaria ischaemum), Trifolium sp. (e.g., Trifolium repens, Trifoliumhirtum, Trifolium incarnatum, Trifolium pratense), Alhagi maurorum,Astragalus spp., Medicago sp. (e.g. Medicago lupulina, Medicagopolymorpha), Melilotus sp., Sesbania sp. (e.g. Sesbania punicea,Sesbania exaltata), Vicia sp. (e.g. Vicia sativa, Vicia villosa),Gallium sp. (e.g., Gallium aparine), Galinsoga sp. (e.g., Galinsogaaristatula), Cardamine sp. (e.g., Cardamine flexuosa, Cardaminehirsuta), Kochia sp. (e.g., Kochia scoparia), Eleusine sp. (e.g.,Eleusine indica), Portulaca sp. (e.g., Portulaca oleraceae), Plantagosp. (e.g., Plantago lanceolata), Euphorbia sp. (e.g., Euphornia supina,Euphorbia maculate, Euphorbia esula, Euphorbia prostrata), Erodium sp.(e.g., Erodium cicutarium), Sonchus sp., (e.g., Sonchus oleraceus),Lactuca sp. (e.g., Lactuca serriola), Capsella sp. (e.g., Capsellabursa-pastoris), Leptochloa sp. (e.g., Leptochloa fascicularis,Leptochloa virgata), Raphanus sp. (e.g., Raphanus raphanistrum),Calandrinia sp. (e.g., Calandrinia ciliata), Paspalum sp. (e.g.,Paspalum dilatatum), Gnaphalium sp., Cynodon sp. (e.g., Cynodondactylon, Cynodon hirsutus), Polygonum sp. (e.g., Polygonum arenastrum,Polygonum lapathifolium), Avena fatua, Hordeum sp. (e.g., Hordeumleporinum), Urtica sp. (e.g., Urtica urens), Tribulus terrestris,Sisymbrium sp. (e.g., Sisymbrium irio), Cenchrus sp., Salsola sp. (e.g.,Salsola tragus, Salsola kali), Amsinckia sp. (e.g., Amsinckialycopsoides), Ipomoea sp., Claytonia perfoliata, Polypogon sp. (e.g.,Polypogon monspeliensis), Xanthium sp., Hypochaeris radicata, Physalissp., Eragrostis sp., Verbascum sp., Chamomilla suaveolens, Centaurea sp.(e.g., Centaurea solstitialis), Epilobium brachycarpum, Panicum sp.(e.g., Panicum capilare, Panicum dichotomiflorum), Rumex acetosella,Eclipta sp. (e.g., Eclipta alba, Eclipta prostrata), Ludwigia sp.,Urochloa sp. (e.g. Urochloa platyphylla, Urochloa panicoides), Leersiasp., Sesbania sp. (Sesbania herbacea), Rotala sp., Ammania sp.,Alternathera philoxeroides, Commelina sp., Sorghum halepense, Partheniumhysterophorus, Chloris truncata, and species in the Fabaceae family.

The Burkholderia strain, compounds and compositions set forth above mayalso be used as a fungicide. The targeted fungus may be a Fusarium sp.,Botrytis sp., Monilinia sp., Colletotrichum sp, Verticillium sp.;Microphomina sp., Phytophtora sp, Mucor sp., Podosphaera sp . . .Rhizoctonia sp., Peronospora sp., Geotrichum sp., Phoma, andPenicillium. In another most particular embodiment, the bacteria areXanthomonas.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES

The compositions and methods set forth above will be further illustratedin the following, non-limiting Examples. The examples are illustrativeof various embodiments only and do not limit the claimed inventionregarding the materials, conditions, weight ratios, process parametersand the like recited herein.

1. Example 1 Isolation and Identification of the Microbe

1.1 Isolation of the Microorganism

The microbe is isolated using established techniques know to the artfrom a soil sample collected under an evergreen tree at the RinnojiTemple, Nikko, Japan. The isolation is done using potato dextrose agar(PDA) using a procedure described in detail by Lorch et al., 1995. Inthis procedure, the soil sample is first diluted in sterile water, afterwhich it is plated in a solid agar medium such as potato dextrose agar(PDA). The plates are grown at 25° C. for five days, after whichindividual microbial colonies are isolated into separate PDA plates. Theisolated bacterium is gram negative, and it forms round, opaquecream-colored colonies that change to pink and pinkish-brown in colorand mucoid or slimy over time.

1.2. Identification on the Microorganism

The microbe is identified based on gene sequencing using universalbacterial primers to amplify the 16S rRNA region. The following protocolis used: Burkholderia sp A396 is cultured on potato-dextrose agarplates. Growth from a 24 hour-old plate is scraped with a sterile loopand re-suspended in DNA extraction buffer. DNA is extracted using theMoBio Ultra Clean Microbial DNA extraction kit. DNA extract is checkedfor quality/quantity by running 5/11 on a 1% agarose gel.

PCR reactions are set up as follows: 2 μl DNA extract, 5 μl PCR buffer,1 μl dNTPs (10 mM each), 1.25 μl forward primer (27F;5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO:1), 1.25 μl reverse primer (907R;5′-CCGTCAATTCCTTTGAGTTT-3′ (SEQ ID NO:2)) and 0.25 μl Taq enzyme. Thereaction volume is made up to 50 μl using sterile nuclease-free water.The PCR reaction includes an initial denaturation step at 95° C. for 10minutes, followed by 30 cycles of 94° C./30 sec, 57° C./20 sec, 72°C./30 sec, and a final extension step at 72° C. for 10 minutes.

The product's approximate concentration and size is calculated byrunning a 5 μl volume on a 1% agarose gel and comparing the product bandto a mass ladder.

Excess primers, dNTPs and enzyme are removed from the PCR product withthe MoBio PCR clean up kit. The cleaned PCR product as directlysequenced using primers 27F (same as above), 530F(5′-GTGCCAGCCGCCGCGG-3′ (SEQ ID NO:3)), 1114F (5′-GCAACGAGCGCAACCC (SEQID NO:4)) and 1525R (5′-AAGGAGGTGWTCCARCC-3′ (SEQ ID NO:5)), 1100R(5′-GGGTTGCGCTCGTTG-3′ (SEQ ID NO:6)), 519R (5′-GWATTACCGCGGCKGCTG-3′(SEQ ID NO:7).

The 16S rRNA gene sequence of strain A396 is compared with the available16s rRNA gene sequences of representatives of the β-proteobacteria usingBLAST. Strain A395 A396 is closely related to members of theBurkholderia cepacia complex, with 99% or higher similarity to severalisolates of Burkholderia multivorans, Burkholderia vietnamensis, andBurkholderia cepacia. A BLAST search excluding the B. cepacia complex,showed 98% similarity to B. plantarii, B. gladioli and Burkholderia sp.isolates.

A distance tree of results using the neighbor joining method, showedthat A396 is related to Burkholderia multivorans and other Burkholderiacepacia complex isolates. Burkholderia plantarii and Burkholderia glumaegrouped in a separate branch of the tree.

The isolated Burkholderia strain was found to contain the followingsequences:

forward sequence, DNA sequence with 27F primer, 815 nucleotides (SEQ IDNO:8);reverse sequence, 1453 bp, using primers 1525R, 1100R, 519R (SEQ IDNO:9);reverse sequence 824 bp using primer 907R (SEQ NO: 10); forward sequence1152 bp using primer 530F (SEQ ID NO:11); forward sequence 1067 bp using1114F primer (SEQ ID NO:12); reverse sequence 1223 bp using 1525R primer(SEQ NO:13); reverse sequence 1216 bp using 1100R primer (SEQ ID NO:14);reverse sequence 1194 bp using 519R primer (SEQ ID NO:15).

1.3. Proof that Burkholderia A396 does not Belong to Burkholderiacepacia Complex

1.3.1 Molecular Biology Work Using Specific PCR Primers

In order to confirm the identification of Burkholderia A396 asBurkholderia multivorans, additional sequencing of housekeeping genes isperformed. Burkholderia multivorans is a known member of theBurkholderia cepacia complex. Efforts are focused on PCR of recA genes,as described by Mahenthiralingam et al., 2000. The following primers areused: (a) BCR1 and BCR2 set forth in Mahenthiralingam et al., 2000 toconfirm B. cepacia complex match and (b) BCRBM1 and BCRBM2 set forthMahenthiralingam et al, 2000 to confirm B. multivorans match. Aproduct-yielding PCR reaction for the first primer set would confirmthat the microbe belongs to the B. cepacia complex. A product-yieldingPCR reaction for the second primer set would confirm that the microbe isindeed B. multivorans.

No PCR product is obtained for either pair of primers. The performanceof the PCR reaction and primers is tested using Burkholderia multivoransATCC 17616 (positive control) and Pseudomonas fluorescens (negativecontrol). Strong bands are observed both for B. multivorans using bothsets of primers. No bands are observed for Pseudomonas fluorescens. Theresults indicate that A396 is a Burkholderia, but not a member of the B.cepacia complex, and not Burkholderia multivorans. This is alsodemonstrated in a comparative culture experiment in which both A396 anda type culture of B. multivorans are grown side-by-side in a shakeculture, and the growth is monitored daily using optical densitymeasurements at 600 nm. Under the set conditions, the novel species A396grew much faster than the B. multivorans type strain (FIG. 1).

1.3.2 DNA-DNA Hybridization

In order to confirm that isolate A396 is a new species of Burkholderia,a DNA-DNA hybridization experiment with Burkholderia multivorans (theclosest 16S rRNA sequence match) is conducted. Biomass for both A396 andB. multivorans is produced in ISP2 broth, grown over 48 hours at 200rpm/25° C. in Fernbach flasks. The biomass is aseptically harvested bycentrifugation. The broth is decanted and the cell pellet is resuspendedin a 1:1 solution of water: isopropanol. DNA-DNA hybridizationexperiments are performed by the DSMZ, the German Collection ofMicroorganisms and Cell Cultures in Germany. DNA is isolated using aFrench pressure cell (Thermo Spectronic) and is purified bychromatography on hydroxyapatite as described by Cashion et al., 1977.DNA-DNA hybridization is carried out as described by De Ley et al., 1970under consideration of the modifications described by Huss et al., 1983using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with aPeltier thermostatted 6×6 multicell changer and a temperature controllerwith in-situ temperature probe (Varian). DSMZ reported % DNA-DNAsimilarly between A396 and Burkholderia multivorans of 37.4%. Theresults indicate that Burkholderia sp strain A396 does not belong to thespecies Burkholderia multivorans when the recommendations of a thresholdvalue of 70% DNA-DNA similarity for the definition of bacterial speciesby the ad hoc committee (Wayne et al., 1987) are considered.

1.4. Biochemical Profile Using Biolog GN2 Plates

For the carbon source utilization profile, A396 is grown overnight onPotato Dextrose Agar (PDA). The culture is transferred to BUG agar toproduce an adequate culture for Biolog experiments as recommended by themanufacturer (Biolog, Hayward, Calif.).

The biochemical profile of the microorganism is determined byinoculating onto a Biolog GN2 plate and reading the plate after a24-hour incubation using the MicroLog 4-automated microstation system.Identification of the unknown bacteria is attempted by comparing itscarbon utilization pattern with the Microlog 4 Gram negative database.

No clear definitive matches are found to the Biolog profile. The closestmatches all had less than 35% similarity with A396: Pseudomonas spinosa(Burkholderia), Burkholderia cepacia, and Burkholderia pseudomallei. Theresults are shown in Table I.

TABLE 1 Biochemical Profile of A396 Substrate Result Substrate ResultCyclodextrin − L-arabinose − Dextrin − D-arabitol − Glycogen −D-cellobiose − Tween 40 + Erythritol − Tween 80 + D-Fructose −N-acetyl-D- − L-Fucose − Galactoseamine N-acetyl-D- − D-Galactose +/−glucosamine Adonitol − Gentibiose − Succinic Acid Mon-methyl −D-Glucose + ester Acetic acid − m-Inositol − Cis-aconitic acid −D-Lactose − Citric acid − Lactulose − Formic acid + Maltose −D-Galactonic Acid Lactone − D-Mannitol − D-Galacturonic Acid − D-Mannose− D-Gluconic acid − D-Melibiose − D-Glucosaminic acid −β-methyl-D-glucoside − D-Glucuronic Acid − D-Psicose − α-hydroxyburyticacid − D-Raffinose − β-hydroxybutyric acid + L-Rhamonose −γ-hydroxybutyric acid − D-Sorbitol − p-hydroxyphenylacetic acid −Sucrose − Itaconic acid − D-Trehalose + α-keto butyric acid − Turanose −α-keto glutaric acid − Xylitol − α-ket valeric acid − Pyruvic AcidMethyl − esther D,L-Lactic acid − Uridine − Malonic acid − Thymidine −Propionic acid + Phenyethyl-amine − Quinic acid − Putrescine −D-Saccharic acid − 2-aminoethanol − Sebacic acid − 2,3-Butanediol −Succinic Acid + Glycerol +/− Bromosuccinic acid − D,L-a-glycerolphosphate +/− Succinamic acid − α-D-Glucose-1- − phosphate Glucuronamide− D-glucose-6-phosphate + L-alaninamide + γ-amino butyric acid +D-Alanine − Urocanic acid − L-alanine + Inosine − L-alanyl-glycine −L-phenylalanine + L-asparagine + L-proline − L-aspartic acid +/−L-pyroglutamic acid − L-glutamic acid + D-serine − Glycyl-L-Asparticacid − L-serine − Glycyl-L-glutamic acid − L-threonine − L-histidine −D,L-carnitine − Hydroxy-L-proline + L-ornithine − L-leucine —

1.5. Fatty Acid Composition

After incubation for 24 hours at 28° C., a loopful of well-grown cellsare harvested and fatty acid methyl esters are prepared, separated andidentified using the Sherlock Microbial Identification System (MIDI) asdescribed (see Vandamme et al., 1992). The predominant fatty acidspresent in the Burkholderia A396 are as follows: 16:0 (24.4%), cyclo17:0 (7.1%), 16:0 3-OH (4.4%), 14:0 (3.6%), 19:0 ω8c (2.6%) cyclo, 18:0(1.0%). Summed feature 8 (comprising 18:1 ω7c) and summed feature 3(comprising of 16:1 ω7c and 16:1 ω6c) corresponded to 26.2% and 20.2% ofthe total peak area, respectively. Summed feature 2 comprising 12:0ALDE, 16:1 iso I, and 14:0 3-OH) corresponded to 5.8% of the total peakarea while summed feature 5 comprising 18:0 ANTE and 18:2 ω6,9ccorresponded to 0.4%. Other fatty acids detected in A396 in minorquantities included: 13:1 at 12-13 (0.2%), 14:1 ω5c (0.2%), 15:0 3-OH(0.13%), 17:1 ω7c (0.14%), 17:0 (0.15%), 16:0 iso 3-OH (0.2%), 16:0 2-OH(0.8%), 18:1 ω7c 11-methyl (0.15%), and 18:1 2-OH (0.4%).

A comparison of the fatty acid composition of A396 with those of knownmicrobial strains in the MIDI database suggested that the fatty acids inthe novel strain A396 were most similar with those of Burkholderiacenocepacia.

1.6 Resistance to Antibiotics

Antibiotic susceptibility of Burkholderia A396 is tested usingantibiotic disks on Muller-Hinton medium as described in PMLMicrobiological's technical data sheet #535. Results obtained after72-hour incubation at 25° C. are presented in Table 2 below.

TABLE 2 Susceptibility of MBI-206 to various antibiotics. Concentration(ug) Susceptible Tetracycline 30 − Kanamycin 30 +++ Erythromycin 15 −Streptomycin 10 − Penicillin 10 − Ampicillin 10 − Oxytetracycline 30 −Chloramphenicol 30 ++ Ciprofloxacin 5 ++ Gentamicin 10 − Piperacillin100 +++ Cefuroxime 30 − Imipenem 10 +++ Sulphamethoxazole- 23.75/25 ++Trimethoprim +++ very susceptible, ++ susceptible, − resistant

The results indicate that the antibiotic susceptibility spectrum ofBurkholderia A396 is quite different from pathogenic B. cepacia complexstrains. Burkholderia A396 is susceptible to kanamycin, chloramphenicol,ciprofloxacin, piperacillin, imipenem, and a combination ofsulphamethoxazole and trimethoprim. As a comparison, Zhou et al., 2007tested the susceptibility of 2.621 different strains in B. cepaciacomplex isolated from cystic fibrosis patients, and found that only 7%and 5% of all strains were susceptible to imipenem or ciprofloxacin,respectively. They also found 85% of all strains to be resistant tochloramphenicol (15% susceptible), and 95% to be resistant (5%susceptible) to the combination of sulphamethoxazole and trimethoprim.Results of Zhou et al., 2007 are similar to those of Pitt et al., 1996who determined antibiotic resistance among 366 B. cepacia isolates andreported that most of them are resistant to ciprofloxacin, cefuroxime,imipenem, chloramphenicol, tetracycline, and sulphametoxacole.

2. Example 2 Burkholderia sp. as an Herbicide

2.1 Study #1

To confirm the activity found in the initial herbicide screen, an invivo study is conducted using the Amberlite 7 XAD resin extract derivedfrom a 5-day old whole cell broth of the novel Burkholderia species. Thedried crude extract is resuspended in 4% ethanol and 0.2% non-ionicsurfactant (glycosperse) at a concentration of 10 mg/mL, and furtherdiluted to a concentration of 5.0 mg/mL. The two samples are sprayed on4-week old plants of bindweed (Convolvulus arvensis), and the plants arekept under growth lights at 25° C. for 2 weeks, at which point, thephytotoxicity evaluations are performed. In the same study, 2-week oldredroot pigweed plants are sprayed with increasing concentrations of thecrude extract derived from the bacterial culture. The testconcentrations are 1.25, 2.5, 5.0 and 10.0 mg/mL, and the plants areincubated as described above before phytotoxicity evaluations.

Results presented in FIGS. 2 (bindweed) and 3 (pigweed) show thephytotoxic effect of Burkholderia crude extract at differentconcentrations, and they show good herbicidal effect on pigweed even atlow treatment concentrations. Both extract treatments (5 and 10 mg/mL)result in stunting on bindweed.

2.2 Study #2

A novel strain of Burkholderia sp. A396 is grown in an undefined mineralmedium for 5 days (25° C., 200 rpm). The whole cell broth is extractedusing XAD7 resin. The dried crude extract is resuspended in 4% ethanoland 0.2% non-ionic surfactant at a concentration of 10 mg/mL, andfurther diluted to concentrations of 5.0, 2.5, and 1.25 mg/mL. All fourtest solutions are then tested on the following broadleaf and grass weedspecies listed in Table 3:

TABLE 3 Broadleaf and Grass Weed Species Tested Common Name ScientificName Lambsquarter Chenopodium album Horseweed Conyza canadensisCurlydock Rumex crispus Crabgrass Digitaria sanguinalis Bluegrass Poaannua Dandelion Taraxacum officinale Nightshade Solanum nigrum MustardBrassica kaber Mallow Malva neglecta Cocklebur Xanthium pensylvanicumBermuda Grass Cynodon dactylon Foxtail Setaria lutescens SowthistleSonchus oleraceusA solution of 0.2% glycosperse and Roundup at 6 fl oz per gallon rate isused as negative and positive controls, respectively.

All plant species are tested in 4″×4″ plastic pots in three replicates.The untreated control plants are sprayed with the carrier solution (4%Ethanol, 0.2% glycosperse) and the positive control plants with Roundupat a rate corresponding to 6 fl. oz/acre. Treated plants are kept in agreenhouse under 12h light/12h dark conditions. Phytotoxicity data taken22 days after treatment for species #1-8 and 12 days for species #9-12are presented in Tables 5 and 6, respectively. The rating scale for bothtables is shown in Table 4:

TABLE 4 Rating Scale Rating Scale % Control 0 0 1 <10 2 25 3 50 4 75 5100

TABLE 5 Phytotoxicity Data for Species #1-8 Treatment HorseweedLambsquarter Dandelion Curlydock Crabgrass Mustard Nightshade BluegrassUTC 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.0 1.25 mg/mL 0.0 4.7 0.0 0.0 0.0 4.30.0 0.0  2.5 mg/mL 0.7 4.5 0.0 0.0 0.0 4.7 0.0 0.0  5.0 mg/mL 4.3 5.00.0 0.0 0.0 5.0 0.0 0.0 10.0 mg/mL 4.7 5.0 0.0 0.0* 0.0 5.0 1.5 0.0Roundup 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 *stunting that resulted inplants approximately half the size of untreated plants

TABLE 6 Phytotoxicity Data for Species #9-12 Cockle- Bermuda Treatmentbur Foxtail Grass Sowthistle Mallow UTC 0.0 0.7 0.0 0.0 2.8 1.25 mg/mL0.5 0.3 0.3 0.0 2.0  2.5 mg/mL 0.5 0.7 0.5 0.0 2.7  5.0 mg/mL 0.8 0.30.2 0.0 2.2 10.0 mg/mL 0.7 0.7 0.3 0.2 1.7 Roundup 4.7 4.8 4.7 5.0 5.0

Based on the results obtained in these studies, the compounds extractedfrom fermentation broths of the isolated Burkholderia species hadherbicidal activity against several weed species are tested. Of thetwelve species tested, Lambsquarters and mustard are most susceptible,followed by mallow and horseweed. Extract concentration as low as 1.25mg/mL is able to provide almost complete control of Lambsquarters andmustard, whereas higher concentration is required for the mallow andhorseweed.

In a separate experiment, using the same design as described above,systemic activity is tested. A 10 mg/ml crude extract supernatant ofBurkholderia sp. A396 is painted onto first true leaves of Ragweed,Mustard, Nightshade, Crabgrass, Wheat and Barnyard Grass. Seedlings areevaluated 7 days after treatment. Observed symptoms include: burning,warping, bleaching Herbicidal activity is observed in the next leafabove the treated leaf in Ragweed, Mustard and Nightshade. No systemicactivity is observed in the tested grasses. In a second experiment, fivefractions of the same crude extract (10 mg/ml) are evaluated using thesame experimental design as described above. Seedlings of Mustard, Wheatand Crabgrass are treated. Seven and 20 days after treatment, symptomsof herbicidal activity are observed in Mustard from four out of the fivefractions (091113B4F6, 091113B4F7, 091113B4F8 and 091113B4F9) using aC-18 column (Phenomenex Sepra C18-E, 50 μm, 65 Å). Symptoms are observedin the next leaf above the treated leaf. No systemic activity isobserved in the tested grasses.

3. Example 3 Burkholderia sp. as an Insecticide

3.1. Contact Activity Studies

The following assay is used in the initial screening phase to determineif the compounds derived from a culture of the novel Burkholderiaspecies has contact activity against a Lepidopteran pest (larvae). It isfurther used as a tool for the bioassay-guided fractionation todetermine the active fractions and peaks derived from thewhole-cell-broth extract. The test is conducted in individual 1.25 ozplastic cups using either Cabbage looper (Tricoplusia ni) late thirdinstar larvae or Beet Armyworm (Spodoptera exigua) early third instarlarvae. A 1 cm×1 cm piece of solid Beet armyworm diet is placed in thecenter of each cup together with one larvae. A 1 μl aliquot of eachtreatment (whole cell broth or extract from a 5-day-old BurkholderiaA396 culture) is injected on each larvae thorax (dorsal side) using aHamilton Precision Syringe. Each treatment is replicated ten times.Water is used as a negative control treatment and malathion as thepositive control treatment. After injection, each cup is covered withparafilm with an airhole, and the cups are incubated for three days at26° C. Mortality evaluations are done daily, starting 24 hours after thetreatment.

FIGS. 4 and 5 present the results from contact activity tests. Accordingto the results, the filter-sterilized broth from a Burkholderia spculture killed about 40% of all test insects within 3 days. Dilutedbroth (50%) has lower activity, resulting in about 10% control in bothinsects tested.

3.2. Activity Against Larvae Through Feeding

Direct toxicity via feeding is tested using the diet-overlay tests withfollowing 96-well plate assay format using microtiter plates with 200 μlof solid, artificial Beet Armyworm diet in each well. One hundred (100)microliters of each test sample is pipetted on the top of the diet (onesample in each well), and the sample is let dry under flowing air untilthe surface is dry. Each sample (filter-sterilized through a 0.2 micronfilter) is tested in six replicates, and water and a commercial Bt (B.thuringiensis) product are used as negative and positive controls,respectively. One third instar larvae of the test insect (Cabbagelooper-Trichoplusia ni; Beet armyworm—Spodoptera exiqua; DiamondbackMoth—Plutella xylostella) is placed in each well, and the plate iscovered with plastic cover with airholes. The plates with insects areincubated at 26° C. for 6 days with daily mortality evaluations.

FIG. 5 represents data from a diet overlay study with Beet Armyworm(Spodoptera exigua) early third instar larvae tested at four differentbroth concentrations: 1× (100%), ¼× (25%), ⅛× (12.5%), 1/16× (6.125%).The data shows that the undiluted, filter-sterilized broth is able togive 100% control at the end of the 7-day incubation period. Similarcontrol is obtained with a 4-fold dilution of the broth, and in the endof the study, both undiluted and 4-fold diluted broths are comparable toBt used as a positive control. However, the effect of Bt issignificantly faster than that of the Burkholderia broths. Efficacyagainst armyworm larvae is dependent on broth concentration, and the twolowest broth concentrations (12.5% and 6.125%) provided less controlthan the two highest ones. However, the performance of the 12.5%dilution is not much lower than the 25% dilution. The 16-fold dilutionof broth is clearly not efficient enough, and it only provided partial(33%) control of armyworm larvae during this 7-day study. Thecorresponding mortality rates for the same broth dilution used oncabbage loopers and diamondback moth larvae are a little higher with6.125% broth killing 80% and 50% of larvae, respectively.

3.3. In Vitro Activity Against Sucking Insects

Five stinkbug (Euschistus sp.) adults are placed in each 16 oz plasticcontainer lined with a piece of paper towel. A microcentrifuge tubecontaining 2 mL of each test sample (filter sterilized whole broth) iscapped with a cotton ball, and laid down on the bottom of the plasticcontainer. One sunflower seed is placed next to the tube as bait. Waterand a commercial product with a mixture of pyrethrin and PBO at arecommended rate are used as negative and positive controls,respectively. Each container is closed with a lid, and they areincubated at 25° C. for 7 days with daily mortality checks.

Results are presented below in Table 7 and they show about 80% controlof sucking insect (stinkbug) by day 7 in this in vitro system with 50%diluted broth. In this study, the diluted fermentation broth ofBurkholderia A396 is more effective in controlling stinkbugs than thecommercial product used as a positive control. Interestingly, thenon-diluted broth resulted in lower insect control, which might be anindication of antifeedant (feeding inhibition) properties of the activesecondary metabolites produced by this new species of Burkholderia.

TABLE 7 Effect of A396 on Stinkbugs % control % control % controlTreatment (Day 3) (Day 5) (Day 7) A396 undil. broth (1x) 0 0 40 A396broth dil. 50% (0.5x) 20 20 80 Pyrethrin + PBO (pos control) 0 0 40Water (neg control) 0 0 0

4. Example 4 Sucking Insect Test In Vivo

The in vivo efficacy of the filtered whole cell broth is tested in aplant assay with mustard plants and green peach aphid (Myzus persicae)as the test insect. Approximately one-month-old Florida Broadleafmustard (Brassica sp.) plants are sprayed with two differentconcentrations (1× and 0.5×) of the filter sterilized whole cell brothof Burkholderia sp. using a Paasche airbrush. Water and a commercialproduct of avermectin (Avid) are used as negative and positive controls,respectively. The plants are allowed to dry on the benchtop, after whichthey are placed in a 6-cup plastic container with a lid with airholes.Ten aphids at various developmental stages are placed on each testplant, and the plants are incubated under growth lamps for 7 days at 25°C. Daily evaluations for the number of aphids on each plant (summarizedin table Table 8 below) are made and recorded in a notebook.

TABLE 8 In vivo Efficacy of A396 on Green Peach Aphids # live # live #live # live aphids aphids aphids aphids Treatment Day 0 Day 2 Day 4 Day7 A396 undiluted broth (1x) 10 36 88 145 A396 broth diluted (0.5x) 10 47138 217 Avermectin (pos control) 10 0 0 0 Water (neg control) 10 140 364393According to the results, both concentrations of the filter-sterilizedbroth derived from a culture of a novel species of Burkholderia are ableto control the population growth of a sucking insect, M. persicae.

5. Example 5 Nematocidal Activity

5.1 Study #1

To assess the effect of filter-sterilized Burkholderia sp A396 culturebroth on the motility (and subsequent recovery) of juvenile (J2)root-knot nematodes (Meloidogyne incognita VW6), the following test isconducted on 24-well plastic cell-culture plates:

A 300-ul aliquot of each test solution (either 1× or 0.5×filter-sterilized broth) is added into appropriate wells after which,fifteen nematodes dispensed in 10 μl of DI water are added into eachwell, plate is closed with a lid, and incubated at 25° C. for 24 hours.Water and Avid at 20,000× dilution are used as negative and positivecontrols, respectively. Effect of each compound on nematode mobility ischecked after 24 hours by probing each nematode with a needle, and theproportion of immobile nematodes in each treatment is recorded in anotebook using a % scale. To assess the recovery of mobility in eachtreatment, a volume of 200 μl is removed from each well, and theremaining solution in each well is diluted by adding 2 mL of DI water.Plates are again incubated for 24 hours as described above, after whichthe second mobility evaluation is performed.

The results presented in FIG. 6 show the filter-sterilized broth at bothtest concentrations can immobilize the free-living juvenile root-knotnematodes. This effect lasts at least for 24-hours, which suggests thatBurkholderia A396 broth can be used to prevent plants from nematodeinfections.

5.2 Study #2

Materials and Methods

Mini Drench Test: Burkholderia A396 whole cell broth is tested in agreenhouse assay conducted in 45 ml pots. Cucumber seeds cv. Toshka aresown directly into pots filled with a sandy loam soil. Ten days later,pots were each treated with 5 ml of a suspension. Specific amounts usedare shown in Table 9:

TABLE 9 Compounds Burkholderia strain A396 Fosthiazate (Standard, EC150) (positive control) Test species Meloidogyne sp. applied at 3000eggs per mini drench pot (in 2 ml) Test plant Cucumis sativus (cucumbercv. Toschka) Test formulation 100% liquid formulation Testconcentrations 100, 50, 25, 12.5, 6, 3, 1.5 ml/L Test application Drenchapplication

As indicated in Table 9, pots are inoculated with 3000 eggs of M.incognita. Four replicates were prepared for each treatment and rate.The trial was harvested fourteen days after trial application andinoculation. Root galling was assessed according to Zeck's gall index(Zeck, 1971). Phytotoxicity was measured as a reduction of root gallingin comparison to the control. The results are shown in FIGS. 9 and 10.

In Mini Drench Test no. 1 (see FIG. 9), the activity of the treatmentwas very high and a reduction of almost 100% was observed when appliedat a concentration of 100 ml/L Burkholderia A396. Fosthiazate performedas usual (100% control at 20 ppm).

In Mini Drench Test no. 2 (see FIG. 10) a 100% reduction of root gallingwas achieved at the highest concentration of 100 ml/L dropping toapproximately 50% at 1.5 ml/L. Fosthiazate performed as usual (100%control at 20 ppm).

5.3 Study #3

To demonstrate the nematicidal activity of Burkholderia A396, agreenhouse study on cucumber (Cucumis sativus) is performed using awhole cell broth of Burkholderia A396 as the test product to controlroot knot nematodes (Meloidogyne incognita). One cucumber plant per potis planted in soil and grown in a greenhouse under artificial lights at28° C. Each pot with a plant is treated with an aliquot (about 80 mL) ofeither the undiluted test product or a test product diluted to 5% withwater. Each Burkholderia A396 treatment as well as a positive controltreatment with Temik (at a label rate) and a negative control with noadditions consisted of five replicates. Plants are grown in a greenhousefor 60 days, after which each plant was harvested and evaluated forfresh shoot and root weights. Number of nematode eggs in each pot wasrecorded and a parameter indicating the number of eggs per a gram ofroot mass was calculated. Statistical analysis (ANOVA) is performed, andthe statistical differences among treatment means at p<0.1 wascalculated. Data presented in Table 10 below shows that even though notstatistically different from the untreated control, the pots treatedwith A396 whole cell broth contained less nematode eggs than theuntreated control pots. The effect calculated as number of eggs per rootmass is more clear when undiluted broth is used as a treatment.

TABLE 10 The effect of A396 whole cell broth on the cucumber shoot androot weight, total number of M. incognita eggs per pot and the number ofeggs per gram of root mass. shoot fresh root fresh # of eggs/g wt wt #of eggs of root untreated 15.22 b 11.76 bc 67693 a 5252.0 ab A396 5% v/v11.89 b  6.914 c 56084 a 8419.4 a A 396 undiluted 15.66 b 11.09 bc 40463a 3929.2 ab Temik 15 G 5 lb/a 29.54 a 29.74 a 68907 a 2604.4 b LSD at p< 0.1  5.34  6.9879 36509.2 3317.07

6. Example 6 Isolation of Templazole A and B Methods and Materials

The following procedure is used for the purification of Templazole A andB extracted from cell culture of Burkholderia sp (see FIG. 7):

The culture broth derived from the 10-L fermentation Burkholderia (A396)in Hy soy growth medium is extracted with Amberlite XAD-7 resin (Asolkaret al., 2006) by shaking the cell suspension with resin at 225 rpm fortwo hours at room temperature. The resin and cell mass are collected byfiltration through cheesecloth and washed with DI water to remove salts.The resin, cell mass, and cheesecloth are then soaked for 2 h in acetoneafter which the acetone is filtered and dried under vacuum using rotaryevaporator to give the crude extract. The crude extract is thenfractionated by using reversed-phase C18 vacuum liquid chromatography(H₂O/CH₃OH; gradient 90:20 to 0:100%) to give 10 fractions. Thesefractions are then concentrated to dryness using rotary evaporator andthe resulting dry residues are screened for biological activity using 96well plate lettuce seeding assay. The active fractions are thensubjected to reversed phase HPLC (Spectra System P4000 (ThermoScientific) to give pure compounds, which are then screened in abovementioned bioassays to locate/identify the active compounds. To confirmthe identity of the compound, additional spectroscopic data such asLC/MS and NMR is recorded.

The active fraction 4 is purified further by using HPLC C-18 column(Phenomenex, Luna 10u C18(2) 100 A, 250×30), water:acetonitrile gradientsolvent system (0-10 min; 80% aqueous CH₃CN, 10-25 min; 80-65% aqueousCH₃CN, 25-50 min; 65-50% aqueous CH₃CN, 50-60 min; 50-70% CH₃CN, 60-80min; 70-0% aqueous CH₃CN, 80-85 min; 0-20% aqueous CH₃CN) at 8 mL/minflow rate and UV detection of 210 nm, to give templazole B, retentiontime 46.65 min. The other active fraction 6 is also purified using HPLCC-18 column (Phenomenex, Luna 10u C18(2) 100 A, 250×30),water:acetonitrile gradient solvent system (0-10 min; 80% aqueous CH₃CN,10-25 min; 80-60% aqueous CH₃CN, 25-50 min; 60-40% aqueous CH₃CN, 50-60min; 40% CH₃CN, 60-80 min; 40-0% aqueous CH₃CN, 80-85 min; 0-20% aqueousCH₃CN) at 8 mL/min flow rate and UV detection of 210 nm, to givetemplazole A, retention time 70.82 min.

Mass spectroscopy analysis of pure compounds is performed on a ThermoFinnigan LCQ Deca XP Plus electrospray (ESI) instrument using bothpositive and negative ionization modes in a full scan mode (m/z 100-1500Da) on a LCQ DECA XP^(plus) Mass Spectrometer (Thermo Electron Corp.,San Jose, Calif.). Thermo high performance liquid chromatography (HPLC)instrument equipped with Finnigan Surveyor PDA plus detector,autosampler plus, MS pump and a 4.6 mm×100 mm Luna C18 5 μm column(Phenomenex). The solvent system consists of water (solvent A) andacetonitrile (solvent B). The mobile phase begins at 10% solvent B andis linearly increased to 100% solvent B over 20 min and then kept for 4min, and finally returned to 10% solvent B over 3 min and kept for 3min. The flow rate is 0.5 mL/min. The injection volume was 10 μL and thesamples are kept at room temperature in an auto sampler. The compoundsare analyzed by LC-MS utilizing the LC and reversed phasechromatography. Mass spectroscopy analysis of the present compounds isperformed under the following conditions: The flow rate of the nitrogengas was fixed at 30 and 15 arb for the sheath and aux/sweep gas flowrate, respectively. Electrospray ionization was performed with a sprayvoltage set at 5000 V and a capillary voltage at 35.0 V. The capillarytemperature was set at 400° C. The data was analyzed on Xcalibursoftware. The active compound templazole A has a molecular mass of 298and showed m/z ion at 297.34 in negative ionization mode. The LC-MSchromatogram for templazole B suggests a molecular mass of 258 andexhibited m/z ion at 257.74 in negative ionization mode.

¹H, ¹³C and 2D NMR spectra were measured on a Bruker 500 MHz & 600 MHzgradient field spectrometer. The reference is set on the internalstandard tetramethylsilane (TMS, 0.00 ppm).

For structure elucidation of templazole A, the purified compound with amolecular weight 298 is further analyzed using a 500 MHz NMR instrument,and has ¹H NMR δ values at 8.44, 8.74, 8.19, 7.47, 7.31, 3.98, 2.82,2.33, 1.08 and has ¹³C NMR δ values of 163.7, 161.2, 154.8, 136.1,129.4, 125.4, 123.5, 123.3, 121.8, 121.5, 111.8, 104.7, 52.2, 37.3,28.1, 22.7, 22.7. Templazole A has UV absorption bands at 226, 275, 327nm, which suggested the presence of indole and oxazole rings. Themolecular formula, C₁₇H₁₈N₂O₃, was determined by interpretation of ¹H,¹³C NMR and HRESI MS data m/z 299.1396 (M+H)⁺ (Calcd for C₁₇H₁₉N₂O₃,299.1397), which entails a high degree of unsaturation shown by 10double bond equivalents. The ¹³C NMR spectrum revealed signals for all17 carbons, including two methyls, a methoxy, a methylene carbon, analiphatic methine, an ester carbonyl, and eleven aromatic carbons. Thepresence of 3′-substituted indole was revealed from ¹H-¹H COSY and HMBCspectral data. The ¹H-¹H COSY and HMBC also indicated the presence of acarboxylic acid methyl ester group and a —CH₂—CH—(CH₃)₂ side chain. Fromthe detailed analysis of ¹H-¹H COSY, ¹³C, and HMBC data it was derivedthat the compound contained an oxazole nucleus. From the 2D analysis itwas found that the iso-butyl side chain was attached at C-2 position, acarboxylic acid methyl ester at C-4 position and the indole unit at C-5position to give templazole A.

The second herbicidally active compound, templazole B, with a molecularweight 258 is further analyzed using a 500 MHz NMR instrument, and has¹H NMR δ values at 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93 and¹³C NMR values of δ 158.2, 156.3, 155.5, 132.6, 129.5, 129.5, 127.3,121.8, 115.2, 115.2, 41.2, 35.3, 26.7, 21.5, 21.5. The molecularformula, is assigned as C₁₅H₁₈N₂O₂, which is determined byinterpretation of ¹H, ¹³C NMR and mass data. The ¹³C NMR spectrumrevealed signals for all 15 carbons, including two methyls, twomethylene carbons, one aliphatic methine, one amide carbonyl, and ninearomatic carbons. The general nature of the structure was deduced from¹H and ¹³C NMR spectra that showed a para-substituted aromatic ring [δ7.08 (2H, d, J=8.8 Hz), 6.75 (2H, d, J=8.8 Hz), and 132.7, 129.5, 115.2,127.3, 115.2, 129.5]. The ¹H NMR spectrum of this structure togetherwith the ¹H-¹H COSY and HSQC spectra, displayed characteristic signalsfor an isobutyl moiety [δ 0.93 (6H, d, J=6.9 Hz), 2.15 (1H, sept., J=6.9Hz), 2.57 (2H, d, J=6.9 Hz). In addition, an olefinic/aromatic proton at(δ 7.06, s), and a carbonyl carbon group (δ 158.9) were also found inthe ¹H and ¹³C NMR spectra. On inspection of the HMBC spectrum, the H-1′signal in the isobutyl moiety correlated with the olefinic carbon (C-2,δ 156.3), and the olefinic proton H-4 correlated with (C-5, δ 155.5;C-2, 156.3 & C-1″,41.2). The methylene signal at δ 3.75 correlated withC-5, C-4 as well as the C-2″ of the para-substituted aromatic moiety.All these observed correlations suggested the connectivity among theisobutyl, and the para-substituted benzyl moieties for the skeleton ofthe structure as shown. In addition, the carboxamide group is assignedat the para position of the benzyl moiety based on the HMBC correlationfrom the aromatic proton at H-4″& H-6″ position. Thus, based on theabove data, the structure was designated as templazole B.

7. Example 7 Isolation of FR90128

The whole cell broth from the fermentation of Burkholderia sp. in anundefined growth medium is extracted with Amberlite XAD-7 resin (Asolkaret al., 2006) by shaking the cell suspension with resin at 225 rpm fortwo hours at room temperature. The resin and cell mass are collected byfiltration through cheesecloth and washed with DI water to remove salts.The resin, cell mass, and cheesecloth are then soaked for 2 h in acetoneafter which the acetone is filtered and dried under vacuum using rotaryevaporator to give the crude extract. The crude extract is thenfractionated by using reversed-phase C18 vacuum liquid chromatography(H₂O/CH₃OH; gradient 90:20 to 0:100%) to give 10 fractions. Thesefractions are then concentrated to dryness using rotary evaporator andthe resulting dry residues are screened for biological activity usingboth insect bioassay as well as herbicidal bioassay. The activefractions are then subjected to reversed/normal phase HPLC (SpectraSystem P4000; Thermo Scientific) to give pure compounds, which are thenscreened in herbicidal, insecticidal and nematicidal bioassays describedbelow to locate/identify the active compounds. To confirm the identityof the compound, additional spectroscopic data such as LC/MS and NMR isrecorded.

Mass spectroscopy analysis of active peaks is performed on a ThermoFinnigan LCQ Deca XP Plus electrospray (ESI) instrument using bothpositive and negative ionization modes in a full scan mode (m/z 100-1500Da) on a LCQ DECA XP^(plus) Mass Spectrometer (Thermo Electron Corp.,San Jose, Calif.). Thermo high performance liquid chromatography (HPLC)instrument equipped with Finnigan Surveyor PDA plus detector,autosampler plus, MS pump and a 4.6 mm×100 mm Luna C18 5 μm column(Phenomenex). The solvent system consists of water (solvent A) andacetonitrile (solvent B). The mobile phase begins at 10% solvent B andis linearly increased to 100% solvent B over 20 min and then kept for 4min, and finally returned to 10% solvent B over 3 min and kept for 3min. The flow rate is 0.5 mL/min. The injection volume is 10 μL and thesamples are kept at room temperature in an auto sampler. The compoundsare analyzed by LC-MS utilizing the LC and reversed phasechromatography. Mass spectroscopy analysis of the present compounds isperformed under the following conditions: The flow rate of the nitrogengas is fixed at 30 and 15 arb for the sheath and aux/sweep gas flowrate, respectively. Electrospray ionization is performed with a sprayvoltage set at 5000 V and a capillary voltage at 35.0 V. The capillarytemperature is set at 400° C. The data is analyzed on Xcalibur software.Based on the LC-MS analysis, the active insecticidal compound fromfraction 5 has a molecular mass of 540 in negative ionization mode.

For structure elucidation, the purified insecticidal compound fromfraction 5 with molecular weight 540 is further analyzed using a 500 MHzNMR instrument, and has ¹H NMR values at 6.22, 5.81, 5.69, 5.66, 5.65,4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23, 1.74,1.15, 1.12, 1.05, 1.02; and has ¹³C NMR values of 172.99, 172.93,169.57, 169.23, 167.59, 130.74, 130.12, 129.93, 128.32, 73.49, 62.95,59.42, 57.73, 38.39, 38.00, 35.49, 30.90, 30.36, 29.26, 18.59, 18.38,18.09, 17.93, 12.51. The NMR data indicates that the compound containsamino, ester, carboxylic acid, aliphatic methyl, ethyl, methylene,oxymethylene, methine, oxymethine and sulfur groups. The detailed 1D and2D NMR analysis confirms the structure for the compound as FR90128 as aknown compound.

8. Example 8 Herbicidal Activity of FR90128

The herbicidal activity of the active compound FR90128 (MW 540) istested in a laboratory assay using one-week old barnyard grass(Echinochloa crus-galla) seedlings in a 96-well plate platform. Onegrass seedling was placed in each of the wells containing 99 microlitersof DI water. One microliter aliquot of the pure compound in ethanol (10mg/mL) is added into each well, and the plate is sealed with a lid. Onemicroliter of ethanol in 99 microliters of water is used as a negativecontrol. The treatments were done in eight replicates, and the sealedplate is incubated in a greenhouse under artificial lights (12 hrlight/dark cycle). After five days, the results are read. The grassseedlings in all eight wells that received the active compound are deadwith no green tissue left, whereas the seedlings in the negative controlwells were actively growing.

9. Example 9 Insecticidal Activity of FR90128

The insecticidal activity of the active compound FR90128 (MW 540) istested in a laboratory assay using a contact bioassay system. Thecompound is dissolved in 100% ethanol to concentrations of 0.001, 0.005,0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 μg/μL. Individual early 3^(rd)instar Beet Armyworm, Spodoptera exigua, larvae are placed in 1.25 ounceplastic cups with a 1 cm² piece of artificial diet (Bio-Serv). AHamilton Micropipette is used to apply 1 μL of compound to the thorax ofeach larvae. Cups are covered with stretched parafilm and a single holeis cut into the parafilm for aeration. Ten larvae per concentration aretreated. The assay is incubated at 25° C., 12 h light/12 h dark. Larvaeare scored at 48 and 72 hours after application. Probit analysis isperformed to assess LC₅₀ value which is found for compound (MW 540) as0.213.

10. Example 10 Isolation of Templamide A, B, FR901465 and FR90128Methods and Materials

The following procedure is used for the purification of compoundsextracted from cell culture of Burkholderia sp (see FIG. 7):

The culture broth derived from the 10-L fermentation Burkholderia (A396)in Hy soy growth medium is extracted with Amberlite XAD-7 resin (Asolkaret al., 2006) by shaking the cell suspension with resin at 225 rpm fortwo hours at room temperature. The resin and cell mass are collected byfiltration through cheesecloth and washed with DI water to remove salts.The resin, cell mass, and cheesecloth are then soaked for 2 h in acetoneafter which the acetone is filtered and dried under vacuum using rotaryevaporator to give the crude extract. The crude extract is thenfractionated by using reversed-phase C18 vacuum liquid chromatography(H₂O/CH₃OH; gradient 90:20 to 0:100%) to give 10 fractions. Thesefractions are then concentrated to dryness using rotary evaporator andthe resulting dry residues are screened for biological activity using 96well plate lettuce seeding (herbicidal) and early 3^(rd) instar BeetArmyworm (insecticidal) assay. The active fractions are then subjectedto repeatedly to reversed phase HPLC separation (Spectra System P4000(Thermo Scientific) to give pure compounds, which are then screened inabove-mentioned bioassays to locate/identify the active compounds. Toconfirm the identity of the compound, additional spectroscopic data suchas LC/MS, HRMS and NMR are recorded.

The active fraction 5 is purified further by using HPLC C-18 column(Phenomenex, Luna 10u C18(2) 100 A, 250×30), water:acetonitrile gradientsolvent system (0-10 min; 80% aqueous CH₃CN, 10-25 min; 80-65% aqueousCH₃CN, 25-50 min; 65-50% aqueous CH₃CN, 50-60 min; 50-70% aqueous CH₃CN,60-80 min; 70-0% aqueous CH₃CN, 80-85 min; 0-20% aqueous CH₃CN) at 8mL/min flow rate and UV detection of 210 nm, to give templamide A,retention time 55.64 min and FR901465, retention time 63.59 min andFR90128, retention time 66.65 min respectively. The other activefraction 6 is also purified using HPLC C-18 column (Phenomenex, Luna 10uC18(2) 100 A, 250×30), water:acetonitrile gradient solvent system (0-10min; 70-60% aqueous CH₃CN, 10-20 min; 60-40% aqueous CH₃CN, 20-50 min;40-15% aqueous CH₃CN, 50-75 min; 15-0% CH₃CN, 75-85 min; 0-70% aqueousCH₃CN) at 8 mL/min flow rate and UV detection of 210 nm, to givetemplamide B, retention time 38.55 min.

Mass spectroscopy analysis of pure compounds is performed on a ThermoFinnigan LCQ Deca XP Plus electrospray (ESI) instrument using bothpositive and negative ionization modes in a full scan mode (m/z 100-1500Da) on a LCQ DECA XP^(plus) Mass Spectrometer (Thermo Electron Corp.,San Jose, Calif.). Thermo high performance liquid chromatography (HPLC)instrument equipped with Finnigan Surveyor PDA plus detector,autosampler plus, MS pump and a 4.6 mm×100 mm Luna C18 5 μm column(Phenomenex) is used. The solvent system consists of water (solvent A)and acetonitrile (solvent B). The mobile phase begins at 10% solvent Band is linearly increased to 100% solvent B over 20 min and then keptfor 4 min, and finally returns to 10% solvent B over 3 min and kept for3 min. The flow rate is 0.5 mL/min. The injection volume is 10 μL andthe samples are kept at room temperature in an auto sampler. Thecompounds are analyzed by LC-MS utilizing the LC and reversed phasechromatography. Mass spectroscopy analysis of the present compounds isperformed under the following conditions: The flow rate of the nitrogengas is fixed at 30 and 15 arb for the sheath and aux/sweep gas flowrate, respectively. Electrospray ionization is performed with a sprayvoltage set at 5000 V and a capillary voltage at 45.0 V. The capillarytemperature is set at 300° C. The data is analyzed on Xcalibur software.The active compound templamide A has a molecular mass of 555 based onthe m/z peak at 556.41 [M+H]⁺ and 578.34 [M+Na]⁺ in positive ionizationmode. The LC-MS analysis in positive mode ionization for templamide Bsuggests a molecular mass of 537 based m/z ions at 538.47 [M+H]⁺ and560.65 [M+Na]⁺. The molecular weight for the compounds FR901465 andFR90128 are assigned as 523 and 540 respectively on the basis of LCMSanalysis. ¹H, ¹³C and 2D NMR spectra are measured on a Bruker 600 MHzgradient field spectrometer.

The reference is set on the internal standard tetramethylsilane (TMS,0.00 ppm).

For structure elucidation of templamide A, the purified compound withmolecular weight 555 is further analyzed using a 600 MHz NMR instrument,and has ¹H NMR δ values at 6.40, 6.39, 6.00, 5.97, 5.67, 5.54, 4.33,3.77, 3.73, 3.70, 3.59, 3.47, 3.41, 2.44, 2.35, 2.26, 1.97, 1.81, 1.76,1.42, 1.37, 1.16, 1.12, 1.04 and has ¹³C NMR values of δ 173.92, 166.06,145.06, 138.76, 135.71, 129.99, 126.20, 123.35, 99.75, 82.20, 78.22,76.69, 71.23, 70.79, 70.48, 69.84, 60.98, 48.84, 36.89, 33.09, 30.63,28.55, 25.88, 20.37, 18.11, 14.90, 12.81, 9.41. The ¹³C NMR spectrumexhibits 28 discrete carbon signals which are attributed to six methyls,four methylene carbons, and thirteen methines including five sp², fourquaternary carbons. The molecular formula, C₂₈H₄₅NO₁₀, is determined byinterpretation of ¹H, ¹³C NMR and HRESI MS data. The detailed analysisof ¹H-¹H COSY, HMBC and HMQC spectral data reveals the followingsubstructures (I-IV) and two isolated methylene & singlet methyl groups.These substructures are connected later using the key HMBC correlationsto give the planer structure for the compound, which has been not yetreported in the literature and designated as templamide A. Thispolyketide molecule contains two tetrahydropyranose rings, and oneconjugated amide.

Substructures I-IV assigned by analysis of 1D & 2D NMR spectroscopicdata.

The (+) ESIMS analysis for the second herbicidal compound, shows m/zions at 538.47 [M+H]⁺ and 560.65 [M+Na]⁺ corresponding to the molecularweight of 537. The molecular formula of C₂₈H₄₃NO₉ is determined byinterpretation of the ESIMS and NMR data analysis. The ¹H and ¹³C NMR ofthis compound is similar to that of templamide A except that a newisolated —CH₂— appear instead of the non-coupled methylene group intemplamide A. The small germinal coupling constant of 4.3 Hz ischaracteristic of the presence of an epoxide methylene group. Thepresence of this epoxide is further confirmed from the ¹³C NMR shiftfrom 60.98 in templamide A to 41.07 in compound with MW 537. Themolecular formulae difference between these two compounds is reasonablyexplained by elimination of the water molecule followed by formation ofepoxide. Thus, on the basis of based NMR and MS analysis the structurefor the new compound was assigned and was designated as templamide B.

For structure elucidation, the purified compound from fraction 5 withmolecular weight 523 is further analyzed using a 600 MHz NMR instrument,and has ¹H NMR δ values at 6.41, 6.40, 6.01, 5.98, 5.68, 5.56, 4.33,3.77, 3.75, 3.72, 3.65, 3.59, 3.55, 3.50, 2.44, 2.26, 2.04, 1.96, 1.81,1.75, 1.37, 1.17, 1.04; and has ¹³C NMR δ values of 172.22, 167.55,144.98, 138.94, 135.84, 130.14, 125.85, 123.37, 99.54, 82.19, 78.28,76.69, 71.31, 70.13, 69.68, 48.83, 42.52, 36.89, 33.11, 30.63, 25.99,21.20, 20.38, 18.14, 14.93, 12.84. The detailed ¹H and ¹³C NMR analysisof compound suggested that this compound was quite similar to compoundtemplamide B; the only difference was in the ester side chain; anacetate moiety was present instead of a propionate moiety in the sidechain. The detailed 1D and 2D NMR analysis confirm the structure for thecompound as FR901465 as a known compound.

Based on the LC-MS analysis, the other compound from fraction 5 has amolecular mass of 540 in negative ionization mode. For structureelucidation, the purified compound from fraction 5 with molecular weight540 is further analyzed using a 500 MHz NMR instrument, and has ¹H NMR δvalues at 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22, 3.21,3.15, 3.10, 2.69, 2.62, 2.26, 2.23, 1.74, 1.15, 1.12, 1.05, 1.02; andhas ¹³C NMR values of 172.99, 172.93, 169.57, 169.23, 167.59, 130.74,130.12, 129.93, 128.32, 73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49,30.90, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51. The NMR dataindicates that the compound contains amino, ester, carboxylic acid,aliphatic methyl, ethyl, methylene, oxymethylene, methine, oxymethineand sulfur groups. The detailed 1D and 2D NMR analysis confirm thestructure for the compound as FR90128 as a known compound.

11. Example 11 Herbicidal Activity of Templamide A, Templamide B,FR901465 and FR90128

The herbicidal activity of templamide A, B, FR901465 and FR90128 aretested in a laboratory assay using one-week old barnyard grass(Echinochloa crus-galla) and lettuce (Lactuca sativa L.) seedlings in a96-well plate platform. One seedling is placed in each of the wellscontaining 99 microliters of DI water. Into each well, a one microliteraliquot of the pure compound in ethanol (10 mg/mL) is added, and theplate is sealed with a lid. One microliter of ethanol in 99 microlitersof water is used as a negative control. The treatments are done in eightreplicates, and the sealed plate is incubated in a greenhouse underartificial lights (12 hr light/dark cycle). After five days, the resultsare read. The grass seedlings in all eight wells that received theactive compound are dead with no green tissue left, whereas theseedlings in the negative control wells are actively growing. Theherbicidal activity of templamide A against lettuce seedlings isslightly lower than for the grass. On the other hand, templamide Bprovides a better (100%) control of lettuce seedlings (used as a modelsystem for broadleaf weeds) than templamide A (Table 11).

TABLE 11 Herbicidal Bioassay data for Templamide A, B, FR901465 andFR90128 Grass seedlings Lettuce seedlings Compounds¹ (% Mortality) (%Mortality) Templamide A 100 88 Templamide B 0 75 FR901465 88 100 FR90128100 88 Control 0 0 ¹10 μg/mL concentration per well

12. Example 12 Insecticidal Activity of Active Compounds

The insecticidal activity of templamide A, B, FR901465 and FR90128 aretested in a laboratory assay using a 96-well diet overlay assay with1^(st) instar Beet Armyworm larvae using microtiter plates with 200 μlof solid, artificial Beet Armyworm diet in each well. One hundred (100)μl of each test sample is pipetted on the top of the diet (one sample ineach well), and the sample is let dry under flowing air until thesurface is dry. Each sample was tested in six replicates, and water anda commercial Bt (B. thuringiensis) product are used as negative andpositive controls, respectively. One first instar larvae of the testinsect (Beet armyworm—Spodoptera exiqua) was placed in each well, andthe plate was covered with plastic cover with airholes. The plates withinsects were incubated at 26° C. for 6 days with daily mortalityevaluations. Based on the results presented in Table 12, templamide Aand B results in 40% and 80% mortality, respectively.

TABLE 12 Insecticidal Bioassay data for Templamide A, B, FR901465 andFR90128 against 1^(st) instar Beet Army Worm (Spodoptera exigua).Compounds¹ (% Mortality) Templamide A 40 Templamide B 80 FR901465 50FR90128 90 Bt 100 Control 0 ¹10 μg/mL concentration per well

Example 13 Fungicidal Activity of FR90128 (MW 540)

Fungicidal activity of FR90128 (MW 540) against three plant pathogenicfungi (Botrytis cinerea, Phytophtora sp., Monilinia fructicola) istested in an in vitro PDA (potato dextrose agar) plate assay. Plates areinoculated with the fungus using a plug method. After the fungus hadestablished and started to grow on the growth medium, eight sterilefilter paper disks are placed on each plate about 1 cm from the edge ina circle. Ten microliters of ethanol solution containing 20, 15, 10,7.5, 5, 2.5 1.25 mg FR90128/mL is added into filter paper disks, and thesolution is left to evaporate. One disk imbedded with 10 μL of pureethanol is used as a negative control. The assay is done with threereplicates. Plates are incubated at room temperature for 5 days, afterwhich the fungicidal activity is recorded by measuring the inhibitionzone around each filter paper disk corresponding to differentconcentrations of FR90128. According to the results, FR90128 has noeffect on the growth of Monilinia but it is effective in controlling thehyphal growth of both Botrytis and Phytophtora. There seems to be aclear dose-response in inhibition with threshold concentrations of 10mg/mL and 1.25 mg/mL for Botrytis and Phytophtora, respectively (FIG.8).

Example 14 Herbicidal Effect of Burkholderia sp. A396 Formulations(Pre-Emergent)

To begin to describe the spectrum of pre-emergence activity, tests wereconducted in petri dish or small pot conditions. In laboratory testing,35 seeds were placed on a ring of blotter paper inside a 3 cm petri dishand supplied with 4 ml of MBI-010 (0.1 mg MBI-005/ml). Water was used asa negative control and oryzalin applied as a positive control. Petridishes were randomly placed in a growth room at 25° C. and 50% RH.Treatments were replicated three times and germinated seeds were counted7 and 14 days after application; water was added as necessary tomaintain moisture levels inside each petri dish.

In pot testing, potting soil was placed into 4 inch square pot, intowhich were then inserted five weed tubers, rhizomes or other undergroundperennation structure, according to species. Pots were drenched with 20mL MBI-010 at a range of dilutions with water. Treatments, includingwater as the negative control and glyphosate as the positive control,were replicated five times. Treatments were evaluated visually as numberof germinating plants per pot and above-ground fresh weights percontainer were taken.

Results in Table 14 indicate broad spectrum activity on both annualgrasses and broadleaves, as well as on some perennials.

TABLE 14A Pre-Emergent Effect of Burkholderia sp. A396 FormulationsPre-Emergent 010 Species Species Plant (common (scientific Scale ProductCategory name) name) Rating (lab/GH/field) Embodiment Grass, annualCrabgrass Digitaria ++++ petri dish Supernatant sanguinalisBarnyardgrass Echionochloa ++++ petri dish Supernatant crus-galliRyegrass Lolium ++++ petri dish Supernatant perenne Late WatergrassEchinochloa + petri dish Supernatant (R) phyllopogon Broadleaf, MustardBrassica kaber ++++ petri dish Supernatant annual Crimson CloverTrifolium ++++ petri dish Supernatant repens Horseweed (R) Conyza ++++petri dish Supernatant canadensis Palmer pigweed Amaranthus ++++ petridish Supernatant (R) palmerii Sdges, Smallflower Cyperus ++++ petri dishSupernatant annual difformis Broadleaf, Field Bindweed Convolvulus ++++pots Supernatant perennial (root segments) arvensis Sedge, PupleNutsedge Cyperus + pots Supernatant perennial (tubers) rotundus RatingScale Rating Pre % Germinaton 0 No Effect  95-100 + Poor 41-95 ++ Fair16-40 +++ Good  6-15 ++++ Great 0-5 S systemic

Example 15 Herbicidal Effect of Burkholderia sp. A396 Formulations(Post-Emergent)

To begin to describe the spectrum of post-emergence activity, tests wereconducted in laboratory and field conditions. For laboratory foliarapplications, 3-10 plants (depending on the species) at the 1-2 leafstage in 2.5 cm square pots containing potting soil were sprayed withMBI-010 at a rate of 40 gal/A using a cabinet track sprayer. Negativecontrols were sprayed with water and positive controls with glufosinate.Pots were randomly placed in a growth room at 25° C. and 50% RH, andwatered as necessary. Treatments were replicated five times andevaluated at 7 and 14 days for visual % damage, with 0% indicating nodamage and 100% indicating plant death.

In drench testing, potting soil was placed into 4 inch square potscontaining plants at the 2-3 leaf stage. Pots were drenched with 20 mLMBI-010 at a range of dilutions with water. Treatments, including wateras the negative control and oryzalin as the positive control, werereplicated five times and kept in a growth room as described above.Treatments were evaluated visually on a percent control basis andabove-ground fresh weights per container were taken.

In field testing, field soil containing weeds at the 1-5 leaf stage weretreated with 50% 010+ water solutions delivered via a hand sprayer tofull coverage. Treatments, including water as the negative control andglufosinate as the positive control, were replicated 3 times and appliedtwice at a four week interval. Treatments were assessed for % control.

Results in Table 15 indicate broad spectrum post-emergence activity onbroadleaves, with little to no activity on grasses, either applied as asoil drench or as a foliar application.

TABLE 15A Post-Emergent Effect of Burkholderia sp. A396 Formulations. AnS indicates an assay that successfully showed systemic activity, a 0indicates no activity. Species Species Plant (common (scientific TestScale Product Category name) name) Mode Rating (lab/GH/field) EmbodimentGrass, Crabgrass Digitaria Foliar ++++ Greenhouse Prototype annualsanguinalis formulation Crabgrass Digitaria Drench 0 GreenhousePrototype sanguinalis formulation Barnyardgrass Echinochloa Foliar +Greenhouse Supernatant crus-galli Barnyardgrass Echinochloa Drench 0Greenhouse Supernatant crus-galli Bluegrass Poa annua Foliar 0Greenhouse CE Broadleaf, Mustard Brassica Foliar ++++ GreenhouseSupernatant annual kaber Mustard Brassica Drench +++ GreenhouseSupernatant kaber Clover Trifolium Drench ++++ Greenhouse Supernatantrepens Lambsquarters Chenopodium Drench ++++ Greenhouse Supernatantalbum Pigweed Amaranthus Spot +++ Greenhouse CE retroflexus PigweedAmaranthus Foliar ++++ Greenhouse Supernatant retroflexus RagweedAmbrosia Foliar S Greenhouse WCB artemisifolia Black Solanum Spot SGreenhouse WCB nightshade nigrum Horseweed Conyza Foliar ++++ GreenhouseCE canadensis Yellow Centaurea Field 0 Field Supernatant Starthistlesolstitialis Mallow Malva spp. Field ++ Field Supernatant Shepherd'sCapsella Field ++ Field Supernatant Purse bursa- pastora Henbit LamiumField 0 Field Supernatant amplexicuale California Medicago Field +++Field Supernatant burclover polymorpha Cutleaf Geranium Field ++ FieldSupernatant geranium dissectum Broadleaf, Dandelion Taraxacum Foliar ++Greenhouse Supernatant perennial oficinale Dandelion Taraxacum Drench 0Greenhouse Supernatant oficinale Dandelion Taraxacum Drench & +++Greenhouse Supernatant oficinale Foliar Bindweed Convolvulus Foliar SGreenhouse WCB arvensis Curly Dock Rumex Foliar ++ Greenhouse CE crispusCrops Fava Beans Foliar ++++ Greenhouse WCB Snap Peas Foliar ++Greenhouse WCB Cucumber Foliar ++++ Greenhouse WCB Radish Foliar ++++Greenhouse WCB Tomato Foliar ++++ Greenhouse WCB Bean Foliar ++Greenhouse WCB Rice Foliar 0 Greenhouse CE Wheat Foliar 0 Greenhouse CESorghum Foliar 0 Greenhouse CE Broccoli Foliar 0 Greenhouse CE PeppersCapsicum Drench 0 Greenhouse Supernatant annum Corn Zea mays Foliar 0Greenhouse CE (conventional) Libert Link Zea mays Foliar 0 Greenhouse CECorn Peanuts Arachis Foliar + Greenhouse Supernatant hypogaea Post ScaleRating % control Rating 0 0 No effect +  1-50 Poor ++ 51-80 Fair +++81-90 Good ++++ 91-100 Great S systemic Systemic CE is concentratedextract; WCB is whole cell broth and Prototype Formulation is whoclecell broth with added surfactants (e.g. hostaphat or genapol)

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Culture Collection(NRRL), 1815 N. University Street, Peoria, Ill. 61604 USA, and given thefollowing number:

Deposit Accession Number Date of Deposit Burkholderia sp. A396 NRRLB-50319 Sep. 15, 2009

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

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1-21. (canceled)
 22. A method for inhibiting infestation of Diptera in alocation where inhibition is desired comprising applying an effectiveamount of a fermented whole cell broth collected from Burkholderia sp.,and at least one of a carrier, diluent or adjuvant, to modulate saidinfestation of Diptera at said location.
 23. The method according toclaim 22, wherein said Burkholderia sp. is Burkholderia A396 strain. 24.The method according to claim 22, wherein said Diptera comprises Aedesspp. or Culex spp.
 25. The method according to claim 24 wherein saidAedes spp. comprises Aedes aegypti or Aedes albopictus.
 26. The methodaccording to claim 22, wherein said Diptera is inhibited by increasingthe mortality of said Diptera.
 27. The method according to claim 22,wherein the mortality of Diptera is increased and wherein there is amortality of Diptera of at least about 50% at said location.
 28. Themethod according to claim 22, which further comprises applying anothernatural or artificial insecticidal substance.
 29. The method of claim28, wherein said another natural or artificial insecticidal substance isapplied at said location in rotation.
 30. The method according to claim22, wherein the infestation of Diptera is inhibited by decreasing therate of hatching of eggs.